In-Vivo and In-Vitro Investigation of Germination Rate of Buried Sclerotia, and Variability in Carpogenic Germination Among Sclerotinia sclerotiorum Isolates

Abstract

The sclerotia of Sclerotinia sclerotiorum serve as a primary inoculum source for initiating infections. This study aimed to evaluate the effects of environmental factors on sclerotial germination under field conditions by establishing sclerotia depots to monitor apothecia appearance over four consecutive years. Additionally, the effects of soil moisture content (25%, 50%, 75%, and 95%), air temperature (10 °C/10 °C, 14 °C/10 °C, and 18 °C/10 °C), and light conditions (white and UV light) on sclerotial germination and apothecial formation were investigated under controlled conditions with a 17 h day/7 h night regime. Furthermore, variability in carpogenic germination among S. sclerotiorum isolates was examined. From 2021 to 2024, significant differences were observed in both the sclerotia germination rate and timing of germination within the season. High soil moisture, particularly prolonged wetness, and soil temperatures between 10 and 14 °C were key factors for apothecial formation under field conditions. Under controlled conditions, higher soil moisture levels (75% and 95%) accelerated sclerotial germination, with sclerotia incubated at 14 °C/10 °C germinating earlier after 38 days than those at 10 °C/10 °C or 18 °C/10 °C. Additionally, the type of light significantly affected apothecial formation, which was observed only in treatments exposed to a combination of white and UV light. Furthermore, significant variations were also found in the duration until sclerotia of different S. sclerotiorum isolates produced the first stipe and the first apothecium, indicating that the genetic characteristics of each isolate affect carpogenic germination.

1. Introduction

Sclerotinia sclerotiorum (Lib.) de Bary is an important necrotrophic soil-borne pathogen with a broad host range in several plant families, such as Asteraceae, Brassicaceae, Chenopodiaceae, Fabaceae, and Leguminosae [1,2,3,4]. The pathogen can attack all parts of the host plant, including the leaves, flowers, fruits, pods, and stems, inducing Sclerotinia stem rot, Sclerotinia head rot, or white mold diseases. These diseases can spread through plants during the vegetation period or at the post-harvest stage, causing severe losses to economically important crops such as soybean, potato, mustard, oilseed rape, and sunflower [5,6,7].

Winter oilseed rape (Brassica napus L.) is a significant arable crop in Germany, covering more than one million hectares per year. Sclerotinia sclerotiorum is among the most economically damaging pathogens affecting oilseed rape cultivation, causing Sclerotinia stem rot. The optimal timing for plant infection occurs during the oilseed flowering stages (BBCH 60–69), typically during late April and early May. Symptoms of Sclerotinia stem rot include the formation and development of bleached, greyish lesions on the main stem, side branches, or pods of the oilseed plant. Additionally, infected stems exhibit the presence of hard, melanized, black sclerotia within the cortex. Other symptoms include early flowering and the premature wilting of plant organs in the terminal parts of the affected stems [8].

The economic importance of winter oilseed rape highlights the need for effective disease management strategies. Crop rotation is limited due to the pathogen’s extensive host range and the prolonged survival of sclerotia in soil. Agronomic practices like cultivar selection, irrigation management, and plant density can reduce disease severity. However, the most effective control strategy currently available is timely fungicide application during the flowering stages. Statistical and mathematical models based on an understanding of the spatio-temporal aspects of disease epidemics integrated into Decision Support Systems (DSSs) aid in the management of Sclerotinia stem rot through timely fungicide applications.

The life cycle of S. sclerotiorum begins with its dormant structures, sclerotia, which survive in the soil for three to seven years [9,10]. Although sclerotia can germinate, giving rise to infective hyphae, myceliogenic germination typically exerts a limited impact on disease epidemiology. In contrast, sclerotia more commonly undergo carpogenic germination, forming apothecia that release airborne ascospores. These ascospores are generally recognized as the primary source of inoculum for Sclerotinia disease epidemics. Consequently, extensive research has been conducted on factors influencing carpogenic germination to predict the production of apothecia and the release of ascospores [11,12,13]. Several environmental factors have been identified as affecting apothecia production. For example, continuous high soil moisture is essential for apothecial development, and even slight moisture tension prevents apothecial formation [3]. Nordin et al. [14] observed that high rainfall during the summer positively correlated with apothecium formation and ascospore release, leading to high Sclerotinia stem rot incidence in oilseed crops in Sweden. They used sclerotia depots to monitor ascospore release periods and estimate Sclerotinia stem rot infection risk. Moreover, the optimal temperature for the carpogenic germination of S. sclerotiorum typically falls between 10 and 20 °C, although there is variation in specific temperature requirements among different laboratories [11,15,16].

Furthermore, the preconditioning of S. sclerotiorum sclerotia has been widely reported as necessary under wet and low-temperature conditions, but the preconditioning methods vary. Some studies advocate for preconditioning at 0 to 5 °C [11,13], while others recommend 8 to 16 °C [11,15] and some conclude that chilling does not enhance carpogenic germination [11,12]. Moreover, wavelengths of light below 390 nm are necessary for stipes to develop into fully expanded apothecia, though this is not a requirement for sclerotia to form stipes [17,18].

Subsequent research has revealed that the duration necessary for the carpogenic germination of S. sclerotiorum isolates is influenced by both the genetic and geographical origin of the isolate. The time needed for apothecium production varies significantly across various studies, spanning approximately 1 to 6 weeks, depending on the preconditioning treatments [12,15]. Nevertheless, there is limited information available regarding the impact of variations in carpogenic germination among distinct isolates of S. sclerotiorum originating from diverse geographical regions.

Between 2020 and 2022, a comprehensive national survey was conducted in major oilseed rape cultivation areas in Germany to assess the incidence of Sclerotinia stem rot [7]. The observed incidence of Sclerotinia disease exhibited significant variations within Germany. Over this period, numerous samples of sclerotia were collected, resulting in the isolation and purification of 62 strains of S. sclerotiorum. Subsequent analysis of these isolates revealed significant variability in both cultural and morphological traits, as well as diverse virulence levels and pathogenic tendencies toward various oilseed rape cultivars [7]. Nevertheless, information regarding differences in carpogenic germination among isolates remains unclear.

The primary objectives of this study were to evaluate the effects of environmental factors on sclerotial germination rates. This was achieved by establishing sclerotia depots at the Julius Kühn-Institute (JKI) campus in Braunschweig, thereby creating a structured foundation for continuously monitoring sclerotial germination under field conditions throughout four consecutive growing seasons. Additionally, the effects of soil moisture, temperature, and UV light on sclerotial germination and apothecial formation were investigated under controlled conditions. Furthermore, this study aimed to explore the variability in carpogenic germination among S. sclerotiorum isolates collected from diverse regions across Germany at various time points.

2. Materials and Methods

2.1. S. sclerotiorum Isolates and Production of Sclerotia

Sixty-two isolates of S. sclerotiorum, collected from naturally infected commercial oilseed rape fields in various regions of Germany between 2020 and 2022 [7] were used in this study (Figure 1). Stocks of each isolate were preserved as sclerotia kept at 4 °C, and new cultures were initiated by placing a sclerotium on potato dextrose agar (PDA; Carl Roth GmbH, Karlsruhe, Germany) and incubating this at room temperature for four days to produce actively growing cultures for inoculum preparation.

To obtain sclerotia for field depots and carpogenic germination tests, sclerotia from each isolate were propagated on an oatmeal sand medium following the method described by Zhou and Boland [19], with some modifications. Briefly, 200 g of quartz sand (<1 mm), 20 g of oatmeal, and 30 mL of demineralized H₂O were added to a 500 mL Erlenmeyer flask, which was then autoclaved at 121 °C for 40 min twice, 24 h apart. Several Sclerotinia mycelial plugs from the margin of new PDA colonies of each isolate were subsequently transferred into each flask. The Erlenmeyer flasks were stored at room temperature for six weeks, during which they were shaken once or twice a week to ensure the formation of uniform sclerotia and to prevent clumping of oatmeal medium and mycelium while incubating. The sclerotia of each isolate were collected from the medium with forceps, dried in an airflow chamber overnight, and stored in sterile Falcon tubes at 4 °C for further use.

2.2. Establishing Sclerotia Depots and Apothecial Emergence Observation

Four sclerotia depots were established near an experimental oilseed rape field on the JKI campus in Braunschweig in late the October period of 2020, 2021, 2022, and 2023. These depots were positioned approximately 500 m apart from each other and consisted of 100 sclerotia, weighing 8–12 mg each [4], which were buried at a depth of 3 cm in grids laid over the soil (Figure 1). The depots remained in the soil throughout the project years. The depots were monitored from April to mid-June of the following year, two to three times a week, and the number of apothecia per depot was counted (Figure 2). Environmental conditions were recorded every 30 min using a data logger (Tinytag Plus 2-TGP-4020; Gemini Data Loggers Ltd., Chichester, UK), with probes recording soil temperature and rainfall.

2.3. Controlled Environment Studies on Carpogenic Germination of Sclerotinia sclerotiorum

Before commencing the main experiment to evaluate the variability in carpogenic germination among various S. sclerotiorum isolates, a series of laboratory experiments was conducted to determine the optimal conditions for the germination of S. sclerotiorum sclerotia and the development of apothecia under controlled conditions.

2.3.1. Assessment of Temperature and Soil Moisture on Sclerotial Germination

Based on observations of the sclerotia depots and data collected under natural field conditions, as well as various studies evaluating the effects of temperature and soil moisture on sclerotia germination [17,20,21], we decided to evaluate the germination rate of sclerotia at temperatures of ±10 °C, ±13 °C, and ±15 °C, and soil moisture levels of 25%, 50%, 75%, and 95%. To achieve this, 25 sclerotia were evenly buried at a depth of 0.5 cm in 300 g of a potting soil/sand mixture (5:1; FloraSelf^®, Braunschweig, Germany; autoclaved at 121 °C for 20 min) in clear plastic storage boxes (500 mL; Papier Brinkmann GmbH, Münster, Germany) with transparent lids. The initial soil moisture content was maintained at 25% of the soil capacity. These boxes were then sealed and incubated at 4 °C for 4 weeks as a cold conditioning treatment to ensure carpogenic germination [12]. After this period, the soil moisture content was adjusted to 25%, 50%, 75%, and 95% of the soil capacity, and the boxes were incubated at 10 °C/10 °C, 14 °C/10 °C, and 18 °C/10 °C (17 h day/7 h night) in a climate chamber. These conditions correspond to the daily mean temperatures of 10 °C, 13 °C, and 16 °C. During the experiment, the boxes were illuminated with a light intensity of 175-µmol m⁻²s⁻¹. Additionally, a black-light lamp was installed to emit ultraviolet (UV) light (Philips TL-D 36W BLB 1SL/25 black-light lamp, Philips lighting DE, Hamburg, Germany). The soil moisture content was maintained by initially adding an appropriate amount of water and subsequently adjusting the weight of each box with additional water twice a week. There were four replicate boxes, each containing 25 sclerotia, and germination was recorded for up to 180 days.

2.3.2. Examining the Impact of Ultraviolet (UV) Light on Apothecia Development

Similar to the previous experiment, the sclerotia were first preconditioned at 4 °C for four weeks. After this period, the soil moisture content was adjusted to 95% of the soil capacity, and the boxes were incubated at 14 °C/10 °C (17 h day/7 h night) in a climate chamber under either white light only or a combination of white light and UV light. The source of white light was two Philips MASTER TL-D Super 80 58W/835 lamps. The source of UV light was one Philips TL-D 36W BLB 1SL/25 black-light lamp (Philips lighting DE, Hamburg, Germany). There were four replicate boxes, each containing 25 sclerotia for each treatment, and the emergence of stipes and apothecia was periodically monitored over 150 days (Figure 3). The experiment was repeated twice.

2.3.3. Carpogenic Germination and Apothecia Development Among S. sclerotiorum Isolates

The sclerotia of each isolate was propagated on the oatmeal sand medium, as previously described. The sclerotia had an average weight of 8–12 mg and were first preconditioned at 4 °C for four weeks. After this period, the soil moisture content was adjusted to 95% of the soil capacity, and the boxes were incubated at 14 °C/10 °C (17 h day/7 h night) in a climate chamber under the conditions of white light and UV light, as described in the previous experiment. There were four replicate boxes, each containing 25 sclerotia of each isolate, and the emergence of stipes and apothecia was periodically monitored over 150 days. The investigation focused on the number of days until the initial stipe emerged and the duration until the first apothecium appeared (Figure 3). The experiment was repeated twice.

2.4. Statistical Analysis

The total sclerotial germination from each temperature–moisture treatment and their interaction were analyzed using a two-factor analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test, deemed significant at a threshold of p ≤ 0.05, using Statistica version 9.1 (Stat Soft, Inc., Tulsa, OK, USA). This analysis determined if the effect of any of the factors was significant. The carpogenic germination and apothecia development among S. sclerotiorum isolates were analyzed using a one-factor analysis of variance.

3. Results

3.1. Sclerotia Depots and Monitoring Apothecial Emergence

Between 2021 and 2024, there were significant differences in the total precipitation and mean soil temperature from March to June (Figure 4). Consequently, major differences were observed in both the sclerotial germination rate and the timing of germination within the season.

In 2021, there was almost no precipitation or only very low amounts ten days before the onset of the oilseed rape flowering stage (Figure 4). Some rainfall events occurred from the middle to the end of the flowering stages. The maximum rainfall recorded in a single day was 20.7 mm on 29 April 2021. However, several rainfall events occurred after the flowering stage, totaling over 3.5 mm and 9.8 mm, respectively. Overall, the period before and during the early flowering stage had more days with precipitation under 1 mm. In comparison, the late- and post-flowering periods had more days with rain over 1 mm and 20 mm (Figure 4). In this year, the soil temperature ranged from 6.7 to 12.9 °C for almost ten days before flowering to the beginning of the flowering stages. During the flowering period, soil temperatures ranged from 10.6 to 12.0 °C. The soil temperature was slightly lower in the late flowering period than in the early to mid-flowering periods. Twenty days post-flowering, the soil temperatures ranged from 14.2 to 17.6 °C. Subsequently, the soil temperature increased to 25 °C (Figure 4). In 2021, the first apothecium was observed on May 10, following several days of consistent rainfall and an increase in soil temperature to 14–15 °C. Sclerotial germination continued until June 15; however, the germination rate remained low, with up to 18 germinated sclerotia per depot on May 25.

In 2022, two to three days of rainfall occurred during the pre-flowering period, just before the first open flowers became visible (Figure 4). One or two rainfall events occurred from the beginning to the end of the flowering period, with a maximum recorded precipitation of 3.1 mm on April 25. During this year, the post-flowering periods experienced more days with rainfall levels exceeding 2.5 mm, reaching up to 14.3 mm (Figure 4). The daily mean soil temperature before the flowering period was lower in 2022 than in 2021. The soil temperature reached 14 °C shortly before the start of the flowering period, remained constant during the flowering period, and then increased to 26 °C. The first apothecia were observed on April 19, with four to six apothecia per depot. The consistent appearance of apothecia continued after the flowering period, although with a low number of apothecia (2–8 apothecia per depot) (Figure 4). No differences in sclerotial germination were observed between depots established in 2020 and those laid in 2021.

Precipitation before the flowering period (March to April) in 2023 was significantly higher than in 2021 and 2022 (Figure 4). This year also had the highest number of rainy days before flowering. The daily rainfall amounts before the flowering period in 2023 ranged from 2.5 mm to 9.6 mm. There was almost no precipitation or very low amounts during the flowering stages. However, a few precipitation events occurred post-flowering, ranging from 5.7 mm to 17 mm (Figure 4). In 2023, the soil temperature ranged from 7.6 to 14.1 °C for almost 20 days before flowering to the beginning of the flowering stages. During the flowering period, soil temperatures increased from 11.4 to 18.0 °C and then decreased to 12.4 °C. The soil temperature was considerably higher in the late and post-flowering periods than in the early to mid-flowering periods (Figure 4). In 2023, major differences were observed in sclerotial germination compared to 2021 and 2022 (Figure 4). As monitoring began in the first week of April, there were 42–60 apothecia in each depot. Until the beginning of the flowering stages, the number of apothecia per depot ranged from 23 to 87. With the start of the flowering stage and the onset of the dry period, the number of apothecia in each depot decreased to an average of 45 apothecia per depot by April 24. The last apothecia were observed around mid-flowering, with 8–14 apothecia per depot. No additional apothecia were found until the end of the flowering period. With the onset of rainy days after the flowering period, a few apothecia were observed in the depots until the end of May, ranging from two to 15 apothecia per depot (Figure 4). Due to the high germination rate in 2023, major differences were observed between depots from different years. The number of apothecia in the 18-month-old depots (laid in November 2021) was higher than the six-month-old depots (laid in November 2022), which was in turn higher than the 30-month-old depots (laid in November 2020).

In 2024, several precipitation events occurred from the end of March to the beginning of April, with amounts ranging from 4.7 mm to 15.6 mm. Additional rainfall was recorded between April 14 and April 19, just before the first open flowers became visible. During the flowering periods, there was almost no rainfall or only very low amounts. Precipitation during the flowering period in 2024 was significantly less than in the previous years. Post-flowering, a few rainfall events occurred, with precipitation ranging from 1.5 mm to 32 mm. Soil temperatures in 2024 ranged from 8.8 °C to 16.2 °C from the end of March to the beginning of April. Temperatures then decreased significantly to 2.8 °C in mid-April before rising to 7.4 °C just before the flowering period. During the flowering period, soil temperatures fluctuated between 8.1 °C and 18.0 °C. After flowering, soil temperatures remained relatively constant, ranging from 11.9 °C to 17.8 °C (Figure 4). In late March 2024, the first apothecia were observed, ranging from six to 11 per depot. A few apothecia also appeared at the beginning of April, significantly before the flowering stage. By the start of the flowering stage, apothecia numbers in depots ranged from two to 14 per depot. Due to dry conditions throughout the flowering period, no apothecia were observed until after the flowering period. From mid to late May, a few apothecia were observed, ranging from two to five per depot. No differences in sclerotial germination were observed between depots established in different years.

3.2. Carpogenic Germination of S. sclerotiorum Under Controlled Conditions

3.2.1. Effect of Temperature and Soil Moisture on Sclerotial Germination

Soil moisture, temperature, and their interactions significantly influenced the days to the first sclerotial germination (p < 0.05) (

Table 1. Analysis of variance (ANOVA) of carpogenic germination of sclerotia of Sclerotinia sclerotiorum under various soil moisture levels and temperatures.

Independent Variables	d.f.	F	p
Soil moisture	3	58.29	<0.05 *
Temperature	2	19.82	<0.05 *
Soil moisture × Temperature	6	8.83	<0.05 *

d.f.: degrees of freedom; F: the value for comparison with the critical value for significance; p: the level of significance (p-value.); (*) denotes statistical significance at the 0.05 level (p < 0.05).

).

At 25% soil moisture content, the initial germination was observed at 60 and 65 days after incubation at 10/10 °C (daily mean temperature of 10 °C) and 14/10 °C (daily mean temperature of 13 °C), respectively. No sclerotial germination occurred at 18 °C and 25% soil moisture content. The final germination rate of sclerotia incubated at 10/10 °C and 14/10 °C with 25% soil moisture content was less than 25% (Figure 5).

Sclerotial germination was observed at all temperatures when the soil moisture level was 50%. However, the highest germination rates occurred when sclerotia were incubated at 10/10 °C or 14/10 °C. At this moisture level, the highest final sclerotial germination was observed in treatments incubated at 10/10 °C or 14/10 °C (up to 50%).

Higher soil moisture levels resulted in more rapid sclerotial germination. Sclerotia incubated at 75% and 95% soil moisture exhibited the quickest response to initial germination (Figure 5). Specifically, sclerotia incubated at 14/10 °C germinated fastest, after 38 days, compared to those at 10/10 °C or 18/10 °C (daily mean temperature of 16 °C). As soil moisture levels increased, the final germination rate also increased. However, across all soil moisture levels, sclerotia incubated at 18/10 °C had a lower final germination rate than those at other temperatures. The final germination rate for sclerotia incubated at 10/10 °C and 14/10 °C with 75% and 95% soil moisture levels was up to 80%. In all treatments, sclerotial germination began to decline after reaching a peak at 130–139 days of incubation (Figure 5).

3.2.2. Effect of UV Light on Development of Apothecia

Exposure to white light alone or in combination with UV light did not significantly affect (p < 0.05) the germination of buried sclerotia (Figure 6). In both treatments, the first stipes appeared 38 days after burying the sclerotia. By 105 days, a maximum of 64 and 59 stipes were observed in trays where the sclerotia were exposed to white light alone or a combination of white and UV light, respectively. Germination of the sclerotia decreased by 150 days after incubation, resulting in the production of 51–52 stipes in each treatment (Figure 6).

In contrast, the type of light significantly affected the formation and development of apothecia (Figure 6). In treatments exposed only to white light, no apothecia were observed throughout the experiment. The first fully developed apothecium was observed 81 days after incubation in trays exposed to a combination of white and UV light. The formation and development of apothecia continued to increase over time. By the end of the experiment, at 150 days, there were 37 to 46 apothecia in trays exposed to the combination of white and UV light (Figure 6).

3.2.3. Carpogenic Germination and Apothecia Development Among S. sclerotiorum Isolates

Our findings revealed significant variations in the duration until the sclerotia of different S. sclerotiorum isolates, collected in different years, produced the first stipe and the first apothecium (

Table 2. Variability in carpogenic germination and apothecial development among Sclerotinia sclerotiorum isolates.

Isolate Number	Year of Collection	Geographical Origin (Federal State)	Days Until the Appearance of the First Stipe			Days Until the Appearance of the First Apothecium
Scl 030/20	2020	Baden-Württemberg	78	±6	c	140	±13	cd
Scl 036/20	2020	Baden-Württemberg	93	±16	cd	132	±8	cd
Scl 010/21	2021	Baden-Württemberg	60	±17	bc	121	±25	c
Scl 011/21	2021	Baden-Württemberg	50	±22	b	102	±22	b
Scl 012/21	2021	Baden-Württemberg	69	±14	bc	0	±0	a
Scl 013/21	2021	Baden-Württemberg	69	±21	bc	144	±7	d
Scl 014/21	2021	Baden-Württemberg	47	±18	b	95	±14	b
Scl 015/21	2021	Baden-Württemberg	62	±17	bc	117	±15	bc
Scl 013/20	2020	Bavaria	55	±12	b	132	±13	cd
Scl 014/20	2020	Bavaria	107	±12	d	126	±8	c
Scl 003/21	2021	Bavaria	68	±15	bc	120	±13	bc
Scl 004/21	2021	Bavaria	64	±17	bc	143	±5	cd
Scl 005/21	2021	Bavaria	54	±6	b	124	±10	c
Scl 006/22	2022	Bavaria	63	±7	bc	113	±12	bc
Scl 007/22	2022	Bavaria	50	±14	b	102	±16	b
Scl 022/20	2020	Hesse	75	±22	bc	101	±6	b
Scl 023/20	2020	Hesse	75	±6	bc	101	±10	b
Scl 024/20	2020	Hesse	62	±21	bc	136	±4	cd
Scl 041/20	2020	Hesse	46	±2	b	99	±14	b
Scl 017/20	2020	Lower Saxony	71	±6	bc	120	±12	bc
Scl 020/20	2020	Lower Saxony	51	±13	b	108	±5	bc
Scl 021/20	2020	Lower Saxony	79	±3	c	132	±5	cd
Scl 025/20	2020	Lower Saxony	38	±11	b	125	±19	c
Scl 040/20	2020	Lower Saxony	90	±23	cd	120	±6	bc
Scl 008/22	2022	Lower Saxony	71	±13	bc	121	±13	c
Scl 018/20	2020	Mecklenburg Pomerania	72	±12	bc	118	±6	bc
Scl 008/21	2021	Mecklenburg Pomerania	88	±23	cd	144	±7	cd
Scl 009/21	2021	Mecklenburg Pomerania	57	±10	b	126	±17	c
Scl 004/22	2022	Mecklenburg Pomerania	47	±28	b	99	±11	b
Scl 005/22	2022	Mecklenburg Pomerania	64	±22	bc	101	±15	b
Scl 035/20	2020	Northrhine-Westphalia	76	±15	bc	138	±17	cd
Scl 006/21	2021	Northrhine-Westphalia	75	±13	bc	146	±4	d
Scl 001/20	2020	Rhineland Palatinate	84	±30	c	144	±3	d
Scl 002/20	2020	Rhineland Palatinate	72	±9	bc	127	±5	c
Scl 003/20	2020	Rhineland Palatinate	65	±19	bc	123	±13	c
Scl 004/20	2020	Rhineland Palatinate	0	±0	a	0	±0	a
Scl 005/20	2020	Rhineland Palatinate	87	±7	cd	153	±10	d
Scl 006/20	2020	Rhineland Palatinate	80	±12	c	133	±10	cd
Scl 007/20	2020	Rhineland Palatinate	72	±12	bc	127	±8	c
Scl 008/20	2020	Rhineland Palatinate	70	±16	bc	124	±8	c
Scl 009/20	2020	Rhineland Palatinate	63	±8	bc	133	±14	cd
Scl 010/20	2020	Rhineland Palatinate	47	±7	b	107	±12	bc
Scl 011/20	2020	Rhineland Palatinate	58	±3	b	120	±16	bc
Scl 012/20	2020	Rhineland Palatinate	70	±6	bc	121	±27	c
Scl 015/20	2020	Saxony	59	±3	b	88	±6	b
Scl 016/20	2020	Saxony	79	±2	c	134	±13	cd
Scl 019/20	2020	Saxony	83	±8	c	141	±5	cd
Scl 033/20	2020	Saxony-Anhalt	74	±7	bc	125	±4	c
Scl 034/20	2020	Saxony-Anhalt	74	±8	bc	126	±11	c
Scl 039/20	2020	Saxony-Anhalt	79	±18	c	131	±8	cd
Scl 001/21	2021	Saxony-Anhalt	59	±14	b	139	±11	cd
Scl 002/21	2021	Saxony-Anhalt	70	±21	bc	145	±3	d
Scl 007/21	2021	Schleswig Holstein	75	±34	bc	135	±23	cd
Scl 001/22	2022	Schleswig Holstein	55	±12	b	98	±10	b
Scl002/22	2022	Schleswig Holstein	72	±21	bc	107	±13	bc
Scl 003/22	2022	Schleswig Holstein	66	±10	bc	95	±14	b
Scl 026/20	2020	Thuringia	93	±23	cd	125	±15	c
Scl 027/20	2020	Thuringia	84	±13	c	110	±11	bc
Scl 028/20	2020	Thuringia	102	±38	d	125	±3	c
Scl 029/20	2020	Thuringia	110	±24	d	145	±12	d
Scl 037/20	2020	Thuringia	92	±17	cd	137	±28	cd
Scl 038/20	2020	Thuringia	65	±10	bc	120	±6	bc

Four replicate boxes, each with 25 sclerotia, were used for each isolate, and the emergence of stipes and apothecia was monitored over 150 days. The investigation recorded the days until initial stipe emergence and the duration until the first apothecium appeared. The experiment was repeated twice. ±SD: Standard deviation; values (mean ± SD) designated with the same letters are not significantly different (p ≤ 0.05) according to Fisher’s least significant test (LSD).

). Among the 62 S. sclerotiorum isolates tested, the sclerotia of one isolate did not germinate. One isolate produced stipes within 38 to 50 days, seven isolates produced stipes within 51 to 70 days, 24 isolates required 71 to 90 days, and six isolates required 91 to 110 days to produce the first stipe (Table 2). Similarly, the isolates showed differences in the production of the first apothecium. Two isolates produced no apothecia, 17 isolates required 88 to 120 days, 28 isolates needed 121 to 140 days, and the remaining 15 isolates took 141 to 153 days to produce the first apothecium (Table 2). These results indicate that the genetic characteristics of each isolate affect carpogenic germination.

4. Discussion

Sclerotinia stem rot, caused by the fungal plant pathogen Sclerotinia sclerotiorum, is one of the major diseases affecting oilseed rape in Europe. In Germany, over the three years of Sclerotinia stem rot monitoring in oilseed rape fields, disease incidence varied by year and location: prevalence across the federal states was 25%, 13%, and 10% in 2020, 2021, and 2022, respectively [7]. Current disease management primarily relies on cultural practices and the application of chemical plant protection [8,22,23]. However, the timing of fungicide applications remains challenging, as the efficacy of fungicides strongly depends on accurately predicting the timing of sclerotial germination and subsequent ascospore release. To date, temperature and water potential are the major limiting factors. However, wide ranges of optimum and minimum requirements for these parameters have been reported. This underscores the urgent need for more specific research in this area.

Therefore, to our knowledge, our study is one of the few to investigate germination rates of buried sclerotia in natural field conditions across multiple years, as well as under controlled environments, while also examining the variability in carpogenic germination among different S. sclerotiorum isolates.

We observed that the timing of apothecia appearance and number of developed apothecia varied significantly among the experimental years. Furthermore, our observations indicated that the frequency of precipitation, rather than the absolute volume, is a dominant factor influencing the number of germinated sclerotia and the appearance of apothecia. Similarly, Bolton et al. [24] reported that soil moisture is essential for the development and formation of apothecia, with ascospore-initiated diseases directly linked to periods of precipitation or irrigation. Comparable results were also observed in dry bean and canola fields when they were irrigated. The experimental manipulation of irrigation showed a positive correlation between the number of apothecia and the frequency of irrigation [25,26]. Furthermore, Bom and Boland [27] demonstrated that a minimum soil moisture value of 10–15 centibars (moist, but not wet soil) was a crucial predictor of fields with a higher number of apothecia and higher disease incidence (>20%) in canola fields. In contrast, field experiments conducted in soybean and carrot showed no correlation between the number of apothecia and precipitation volumes at 1-week to 2-month intervals preceding assays [24,28,29]. However, whether a field was irrigated was an essential factor in developing predictive models for apothecia appearance in soybean fields [29]. Moreover, soil mineral mobilization is more efficient in wet or humid conditions than in dry conditions, significantly influencing the germination of sclerotia and the formation of apothecia in S. sclerotiorum. In moist environments, the availability of essential nutrients is enhanced, supporting the metabolic processes required for sclerotia to germinate. This increased nutrient availability can also lead to more robust fungal growth and a higher likelihood of successful apothecium formation.

Although several studies under controlled greenhouse or laboratory conditions have indicated a significant correlation between apothecia development and soil temperature and moisture [12,30], few field studies have included soil measurements in their research [29,31]. However, it should be considered that changes in soil temperature are slower and exhibit less diurnal variation compared to air temperature. Furthermore, soil temperature varies with depth. The surface soil temperature can change more rapidly with weather conditions, while deeper layers tend to remain more stable throughout the day. Additionally, soil has a higher heat capacity, meaning it can store more heat and takes longer to heat up and cool down. Finally, the moisture content in soil can significantly influence its temperature, with wet soil heating up and cooling down more slowly than dry soil. In the present study, monitoring depots under field conditions showed that, similar to precipitation and soil moisture, soil temperature plays an essential role in sclerotial germination and apothecial formation. In the years with lower soil moisture (e.g., 2021 and 2022), the first apothecia appeared after a rain event when the soil temperature increased to approximately 14 °C. In 2023, higher soil moisture resulted from frequent rainfalls, and the first apothecia appeared when the soil temperature reached 9 to 10 °C. Higher soil temperatures (>18 °C) had a negative effect on apothecia formation, causing also the existing apothecia to dry out quickly after appearing. No apothecia were found in soil temperatures below 9 °C or over 26 °C. Comparable findings were reported by Huang and Kozub [16], indicating that favorable temperature conditions mostly range from 10 to 20 °C. However, the temperature needs are dependent on the origin of the S. sclerotiorum isolates and the temperature at which the sclerotia are produced. Furthermore, Fall et al. [31], found a strong correlation between the mean temperature and the number of developed apothecia in most experimental plots and years in soybean fields. An in-depth analysis of this association revealed a strong nonlinear pattern, in which apothecia were predominantly found at soil temperatures between 15 and 25 °C, with significant declines in the number of apothecia outside this range. However, in carrot fields, there was no significant correlation between the number of developed apothecia and soil temperature 1, 2, or 3 weeks before apothecia surveys [28].

Another noteworthy result from monitoring depots of different years was that sclerotia could survive for at least four years when buried in non-cultivated soil. However, the age of the sclerotia affects the potential for germination. In 2023, due to the high sclerotial germination rate, significant differences in the number of developed apothecia were observed among depots from different years. The number of apothecia in 18-month-old depots (established in November 2021) was higher than in both 6-month-old depots (established in November 2022) and 30-month-old depots (established in November 2020). Additionally, the number of apothecia in 6-month-old depots exceeded that in 30-month-old depots. This suggests that newly formed sclerotia have a latency phase (dormancy) lasting several weeks or months. Similar results were revealed by Cook et al. [32], who found that more than 50% of the sclerotia survived for at least three years when buried in non-cultivated soil. Cosic et al. [33] described that the percentage of viable sclerotia can be up to 100% after three years. Other studies have shown that sclerotia can survive up to four or five years [34]. Additionally, numerous studies have also demonstrated the effect of burial depth, soil moisture, and soil temperature on sclerotial survival [10,35,36]. Among these factors, flooding appears to be the most detrimental. Matheron and Porchas [35] reported that sclerotia might completely decay within 14–21 days under flooding conditions. Similar results were found by Cosic et al. [33], who observed that continuous flooding entirely destroyed sclerotia buried in the soil at a depth of 5 cm.

The second part of the current study was to evaluate the effects of soil moisture, temperature, and light conditions (white and UV light) on the germination of sclerotia and the formation of apothecia under controlled environments. We showed that temperature, moisture, and their interactions significantly affected the days until the first sclerotia germinated. Those present in a dry environment (soil moisture less than 25%) were unable to germinate carpogenically, especially in 18/10 °C temperature in comparison with 10/10 and 14/10 °C. In contrast, saturated soil (soil moisture more than 75%) enhanced the sclerotial germination, particularly in experiments with air temperatures of 10/10 and 14/10 °C. Similar results were observed by Clarkson et al. [37], who showed that the carpogenic germination of sclerotia occurred between 5 and 25 °C, but only when the soil water potential was more than −100kPa. The final germination rate decreased considerably with temperatures approaching 25 °C in comparison with temperatures of ≤20 °C. In their study, the germination rate of sclerotia correlated positively with temperature, and the final number of germinated sclerotia was a function of temperature fitted with a probit model [37]. Furthermore, Wu and Subbarao [11] reported that under controlled greenhouse conditions, a 10- to 20-day dry period completely inhibited carpogenic germination, whereas maximum carpogenic germination was observed in fully water-saturated soil [38]. In their study, no apothecia developed below 70 to 80% soil water saturation. Variation between results of different studies could be due to factors such as variability between isolates of different geographic origins. For instance, Huang and Kozub [32] described that sclerotia which formed at 10 °C from isolates originating in cool, temperate regions produced apothecia more readily than those which formed at 25 to 30 °C. However, the reasons for this difference and its implications need to be explored further. Furthermore, Sansford and Coley-Smith [39] reported that apothecial production was poor for sclerotia isolates from warmer climatic regions formed at warmer temperatures unless a period of cold conditioning at 10 °C was applied. The mechanisms underlying this phenomenon, along with the potential for developing more effective treatments for managing sclerotia germination, necessitate further investigation. Such a conditioning period is generally required for most S. sclerotiorum isolates to ensure carpogenic germination of sclerotia formed at approximately 15 °C or above, with standard treatments by researchers being either 5 or 10 °C for four weeks. In German oilseed rape fields, sclerotia naturally receive sufficient cold conditioning during the winter, resulting in the appearance of apothecia in spring as soil temperatures rise. In our study, preconditioning sclerotia by incubating them at 4 °C for 4 weeks significantly enhanced their germination.

The third important factor affecting the development of apothecia was the presence of UV light. In the current study, no apothecia developed under white light alone. However, when UV light was added to the white light, the first fully developed apothecia were observed 83 days after sclerotia incubation. The number of developed apothecia continued to increase until the end of the experiment at 150 days after incubation. In a previous study, Sun and Yang [12] quantified the effects of light, soil moisture, and temperature on the apothecium production of S. sclerotiorum. They demonstrated that both light intensity and the soil moisture level influenced the optimal temperature and the temperature range for sclerotial germination. Furthermore, Veluchamy and Rollins [40] reported that upon germination, the apothecial stipe requires exposure to UV-A wavelengths of light to develop a fertile disc. They identified a gene, cry1, from S. sclerotiorum that is closely related to photolyase/cryptochrome proteins in the CRY-DASH family. They further characterized this CRY-DASH ortholog from S. sclerotiorum and observed significant transcript accumulation only after exposure to UV-A, not in response to other wavelengths of light. However, in oilseed rape fields, natural light may affect apothecium production differently than light in climate chambers. It is possible that natural light has a higher intensity and a more significant effect on apothecium production compared to artificial light. Therefore, it is noteworthy to evaluate the effect of row spacing and canopy closure in oilseed rape fields on light intensity at the soil surface and subsequent apothecium development.

The final part of this study aimed to explore the variability in carpogenic germination among S. sclerotiorum isolates collected from diverse regions across Germany at various time points. The evaluation of apothecia production is important, as apothecia resulting from the carpogenic germination of sclerotia serve as the primary source of inoculum (ascospores) in oilseed rape fields. In a previous study, the S. sclerotiorum population used in was found to exhibit wide variability in morphological traits, cultural characteristics, and virulence [7]. Here, we observed significant variations in the time it took for the sclerotia of different S. sclerotiorum isolates to produce the first stipe (ranging from 38 to 110 days) and the first apothecium (ranging from 88 to 153 days). One isolate’s sclerotia did not show carpogenic germination, and two isolates did not produce developed apothecia. This variation was not dependent on the year of isolate collection or their geographic origin. As all isolates were subjected to the same experimental conditions; thus, these differences are likely attributable to genetic variations among the isolates. A previous study in canola fields evaluated 35 isolates of S. sclerotiorum and observed 100% carpogenic germination, although the time required for stipe formation varied considerably [41]. Similarly, Abreu and Souza [42] evaluated 50 isolates of S. sclerotiorum collected from common beans over different years. They characterized the isolates based on their ability and time required to develop apothecia. While most isolates had the ability to form apothecia, the time required for full apothecium development varied among them. Further molecular studies indicated that the FoxE2 gene is required for apothecial development in S. sclerotiorum populations. The Ss-FoxE2 gene is expressed significantly higher in the apothecial stages than in other developmental stages, which may affect the time required for the full development of apothecia. However, although Ss-FoxE2 appears to be necessary for regulating sexual reproduction, it may not significantly affect the pathogenicity and vegetative development of S. sclerotiorum.

5. Conclusions

For effective control of Sclerotinia stem rot, the evaluation of apothecia production is crucial, as apothecia resulting from the carpogenic germination of sclerotia serve as the primary source of inoculum (ascospores) in oilseed rape fields. This study focused on the precipitation and temperature requirements for sclerotial germination before, during, and after the flowering stages of winter oilseed rape. The carpogenic germination of sclerotia was found to depend more on the frequency of precipitation rather than the absolute volume and on optimal temperature conditions. Additionally, the genetic characteristics of each isolate significantly affected the production of stipes and apothecia. However, it should be taken into consideration that beside environmental conditions, the local soil microbiota significantly influences the germination of sclerotia produced by S. sclerotiorum. Beneficial microbes can compete for resources, produce synergistic compounds, and alter nutrient availability, pH, and moisture conditions, all of which affect sclerotia germination. Some soil microbes may even release metabolites that either inhibit or promote germination. Additionally, the structure and biodiversity of the soil microbiome contribute to overall soil health, potentially affecting pathogen dynamics.

These findings have practical implications for agronomic decision-making and the management of Sclerotinia stem rot. Understanding the conditions necessary for the germination of sclerotia and the development of apothecia can help determine the optimal timing for fungicide application. This knowledge allows for more effective and precise use of fungicides, potentially reducing costs and environmental impact. Moreover, it can aid in developing predictive models for disease outbreaks, enabling farmers to implement preventative measures promptly. Future research should focus on the integration of these environmental factors and the genetics of the pathogen into comprehensive management strategies, ensuring sustainable and productive oilseed rape cultivation.