Effect of low temperature culture on the biological characteristics and aggressiveness of Sclerotinia sclerotiorum and Sclerotinia minor

This is anOpe Abstract – Sunflower White Mold caused by Sclerotinia sclerotiorum and Sclerotinia minor is a devastating disease worldwide. To investigate the effect of low temperature (4 °C) on biological characteristics and aggressiveness of isolates of the two species, which were collected from the same field in Baiyinchagan, Inner Mongolia, their mycelial growth rate, oxalic acid secretion level and polygalacturonase activity were compared under normal culture temperature (23 °C) and low temperature (4 °C). Aggressiveness was also evaluated on detached leaves by inoculating the isolates produced in both temperatures. The results suggested that culture of isolates at 4 °C not only promoted mycelial growth, but also enhanced secretion of oxalic acid and polygalacturonase activity of both S. sclerotiorum and S. minor isolates compared to that cultured at 23 °C. Additionally, the corresponding aggressiveness of tested isolates of the two species also increased after culture at 4 °C. However, S. sclerotiorum always showed faster mycelial growth, higher oxalic acid levels and greater polygalacturonase activity than S. minor at both 23 °C and 4 °C, indicating that S. sclerotiorum is generally the more aggressive species than S. minor.


Introduction
Sunflower White Mold (SWM) caused by both Sclerotinia sclerotiorum and Sclerotinia minor is one of the most important diseases of sunflower and can cause serious yield losses (Gulya et al., 1997;Van Becelaere and Miller, 2004;Liu et al., 2018). Inner Mongolia, located in northern China is a major confectionery sunflower producing area, with a planting area around 0.86 million hectares. S. sclerotiorum and S. minor are two necrotrophic pathogens with broad host ranges and distributed globally, although S. sclerotiorum is more common than S. minor in most sunflower planting regions (Wu et al., 2008;Roghayeh et al., 2018). The symptoms of SWM caused by S. sclerotiorum and S. minor are different. S. sclerotiorum causes root, stem, leaf and capitulum rot at different developmental stages of sunflower (Sharma et al., 2016), whereas, S. minor causes only basal stem rot (Li et al., 2016b) .
The infection process of SWM can be divided into two phases: biotrophic and necrotrophic phase (Kabbage et al., 2013). Following a short biotrophic phase after infection, both S. sclerotiorum and S. minor can cause tissue maceration and necrosis, leading to rapid cell death and host cell wall degradation. Toxins and cell wall degrading enzymes (CWDEs) such as polygalacturonase (PG) are believed to play critical roles in promoting these processes (Xia et al., 2020). Previously, oxalic acid (OA) was thought to be a necessary virulence factor for S. sclerotiorum infection, not only reducing the pH of host cells and enhancing the activity of CWDE, but also inhibiting the oxygen burst and selfphagocytosis, thus facilitating cell apoptosis in host plants (Godoy et al., 1990;Guimarães and Stotz 2004;Williams et al., 2011;Kabbage et al., 2013). This is also called the oxalate-dependent theory. However, some data indicated that OA is not a pivotal pathogenic factor, but only provides an acidic environment for pathogen infection, this is known as the pH-dependent theory (Xu et al., 2018). Apart from the pathogenicity theories mentioned above some effectors have been identified based on genomic sequence analysis, and these have been verified as functional virulence factors if the host does not contain corresponding resistance proteins (Liu et al., 2015).
The two pathogens can form sclerotia as dormant structures at late developmental stages of sunflower, but sclerotia formed by S. minor are much smaller than those of S. sclerotiorum (Li et al., 2016a). Environmental factors such as soil temperature and moisture can influence sclerotia formation and mycelial growth rate of both S. minor and S. sclerotiorum. Fernandes et al. (2016) reported that the temprature suitable for mycelium growth of both pathogens was possible between 7°C and 27°C, but 22°C is the most suitable cultural temperature and no mycelia growth at 32°C. The specific temperature and time required for preconditioning of sclerotia to stimulate carpogenic germination vary among different studies (Clarkson et al., 2017;Dillard et al., 1995;Mila et al., 2008).
In this study, low temperature culture (4°C) was found accidently to affect the development of both S. sclerotiorum and S. minor. The mycelial growth rate, oxalate secretion ability and PG enzyme activity were compared at 23°C and 4°C using isolates of both species, which were collected from diseased sunflower plants. The aggressiveness of the isolates tested was also determined using a detached leaf inoculation method. The aim of this study was to make clear the effects of 4°C culture on both the biological characteristics and aggressiveness of S. sclerotiorum and S. minor.
Sclerotia collected from individual infected sunflower stems were surface-sterilized in 75% alcohol for 30 s, then in 0.1% NaCIO for 3 min and finally washed 3 times with distilled water. The sterilized sclerotia was then placed on potato dextrose agar (PDA) and incubated for 72 h at 23°C. Pure cultures were obtained after three hyphal tip multiplications. The sclerotia formed on the edge of the purified colonies were collected and stored at 4°C for further analysis.
The seeds of confectionery sunflower variety (LD5009) were purchased from Beijing Kaifuri Seed Industry Company.

Cultural mediums
Potato dextrose agar (PDA) medium was made from 200 g of potato, 20 g of dextrose, and 15 g of agar per liter of distilled water.
Potato dextrose broth (PDB) medium consisted of 200 g potato, 15 g dextrose per liter of distilled water.

Low-temperature processing method
Purified S. sclerotiorum and S. minor isolates were cultured on PDA plates at 23°C for two days, then 5 mm diameter mycelial plugs were cut from the edge of the colony and transferred on to a new PDA plate. The plates were cultured at either 23°C (control) or 4°C (low temperature). When the colony of the control covered 3/4 of the plate, mycelial plugs of both S. sclerotiorum and S. minor cultured at 4°C were cut from the edge of colonies to perform the following experiments.

Determination of mycelial growth rate
The mycelial plugs (5-mm diameter) cut from the actively growing edge of low temperature culture plate were placed on new PDA plates and cultured under normal condition (23°C). The cross diameter method was used to measure and record the colony diameters of each isolate, with three replications. The average diameter recorded in the table was calculated from the diameter of the three replicates measured after culture for 3 days.

Oxalic acid (OA) measurement
A 5-mm diameter mycelial plug cut from the actively growing edge of a 2-day-old culture colony was inoculated into 40 mL of PDB and incubated in a static position at 23°C for 5 days. The filtrate of the cultur was used for analyzing OA concentration following the procedure described by Durman et al. (2005). The reaction mixture containing 0.2 mL filtrate (or standard oxalic acid solution), 0.11 mL BPB 0.1 mm, 0.20 mL sulfuric acid 1M, 0.18 mL mL potassium dichromate 100 mm and 4.8 mL mL of distilled water was placed in a 60°C water bath for 10 min, followed by quenching and addition of sodium hydroxide solution to the final concentration of 0.75 mm. The absorbance was measured at 600 nm using a spectrophotometer (TU-1901, Beijing Purkinje General liability company, China). OA concentration was calculated based on a standard curve. The assay was performed in triplicates and each with three technical replicates.

Polygalacturonases (PG) enzymatic assay
A 5 mm diameter mycelia plug cut from actively growing edge of colony was inoculated into a flask containing 80 mL PDB. Cultures were shaken at 140 rpm at 23°C for 10 days. After centrifuging at 5000 rpm for 15 min, the aqueous phase was decanted and assayed for PG enzymatic activity. The 3, 5 dinitrosalicylic acid (DNSA) assay was used to assay PG enzymatic activity (Moyo et al., 2003). A 400 mL aliquot of 0.5% pectin was mixed with culture filtrate and topped up to a final volume of 500 mL by adding 0.1 mol/L sodium acetate. Samples were incubated at 30°C for 5 h. DNSA reagent (44 mmol/L DNSA, 4 mmol/L sodium sulphite, 75 mmol/L NaOH) was added and the samples were boiled for 10 min to stop the reaction. The volume of samples was adjusted to 2 mL by adding deionized water and the absorbance was measured at 540 nm using a spectrophotometer. Absorbance values were used to determine the enzymatic activity from a standard curve generated with D-(þ) galacturonic acid. The assay was performed in triplicate and each with three technical replicates.

Aggressiveness assessment
When the colony diameter of control isolates covered 3/4 of the petri dish under 23°C culture condition, the PDA plates cultured under 4°C were taken out of the incubator. A 5 mm diameter mycelia plug was cut from the edge of colony and transferred into MM medium. Aggressiveness assay was performed on detached leaves of confectionery sunflower variety LD5009. The leaves were collected from sunflower seedlings with 6 true leaves (V6 stage) and placed on a moist filter paper soaked in a covered plastic container (Length 32.5 cm Â width 26 cm Â height 11.5 cm).
A 5 mm diameter mycelial plug was cut from the actively growing edge of 3-day-old culture on MM media and placed upside down on the abaxial side of the detached leaves. The MM plain plug (control) was inoculated on the other side of same inoculated leaf as a control. The covered plastic container containing the inoculated leaves were kept at room temperature (RT) and the lesion diameters were measured after 48 h post inoculation (hpi). The pathogenicity assay of each isolate was performed on five detached leaves and the average size of the lesion diameters represented the aggressiveness of each isolate.

Data analysis
Differences in biological characteristics and aggressiveness of cultured isolates under normal and low temperature were analyzed using SPSS 17.0 software. Duncan's new complex range method was used to test the significance of differences under 0.05 level.

Promoting mycelial growth of S. sclerotiorum and S. minor after culture at 4°C
The mycelia growth rate of all tested isolates cultured at 4°C was faster than those cultured at 23°C (Fig. 1). The average diameter of S. sclerotiorum and S. minor cultured under 23°C for two days was 6.95 ± 0.57 cm and 5.02 ± 0.34 cm respectively, whereas, under same culture period at 4°C was 8.07 ± 0.09 cm and 6.69 ± 0.23 cm respectively, indicating that the low temperature accelerated the mycelia growth of isolates of both species used in this study (Tab. 1). However, the average mycelia growth rate of S. sclerotiorum was significantly faster than that of S. minor under both cultural temperature conditions (Tab. 1).

OA secretion and PG enzymes activity of S. sclerotiorum and S. minor after culture at 4°C
The OA secretion ability of both S. sclerotorum and S. minor isolated was observed boosted after culture at 4°C. The average OA value of S. sclerotiorum and S. minor after 4°C culture was 32.73 ± 2.89 mg/mL and 27.32 ± 0.21 mg/mL, compared with 27.69 ± 1.42 mg/mL and 20.63 ± 2.25 mg/mL respectively at 23°C (Tab. 1). The average OA values showed the same pattern as mycelial growth rate, higher for S. sclerotorum than for S. minor at both temperatures. OA levels of S. sclerotiorum isolates S.s-2 and S.s-5 increased from 26.69 ± 0.68 mg/mL and 27.06 ± 1.02 mg/mL at 23°C to 35.84 ± 0.96 mg/mL and 32.24 ± 0.62 mg/mL respectively at 4°C. For S. minor isolates, the same trend was detected, with S.m-8 showing the greatest increase (9.01 mg/mL), followed by S.m-1 (6.94 mg/mL) and S.m-3 (4.12 mg/mL).
The average PG enzymatic activities of S. sclerotiorum and S. minor were 26.37 U/mg and 24.54 U/mg respectively after culture under 4°C, compared with 22.56 U/mg and 21.57 U/mg respectively at 23°C (Tab. 1), with no significant difference between species.
For S. minor, PG enzymatic activity of all three isolates increased significantly, especially for S.m-8, the increased value listed at the top, it is 3.48 U/mg, whereas, for S.m-3 and S.m-1, the increase value is 3.32 U/mg and 2.13 U/mg respectively.
The average lesion diameter of S. sclerotiorum and S. minor cultured at 4°C was 3.34 ± 0.38 cm and 2.43 ± 0.14 cm respectively, while it was 2.57 ± 0.27 cm and 2.08 ± 0.11 cm at 23°C (Tab. 1). The enhanced aggressiveness of S. sclerotiorum at 4°C was more significant than that of S. minor., the two species showed a significant difference.

Conclusion and discussion
In Inner Mongolia, both S. sclerotiorum and S. minor were isolated from diseased plants with SWM symptoms, but, S. minor isolates were only collected from sunflower fields which were located in the northern piedmont region of Yin mountain with high altitudes and low temperatures in summer. The lowest temperature in August is about 6°C. Sometimes, SWM infected plants caused by S. sclerotiorum were also observed in fields in which SWM was dominantly caused by S. minor, but co-infection by both S. sclerotiorum and S. minor was never encountered. Previous studies showed that mycelial growth of both S. sclerotiorum and S. minor occurred at temperatures ranging from 12°C to 27°C, and sclerotia of S. minor germinated and exhibited mycelial growth at temperatures ranging from 6°C to 30°C, but S. minor shows much more sensitivity to low temperature (Domingues et al., 2016). Based on the low temperatures in summer in the region where we observed SWM caused by S. minor, it was speculated that S. minor is favorised by such low temperature. Isolates of S. sclerotiorum collected from regions with mild temperatures do need cold "conditioning" treatment in winter to promote sclerotia germination and producing apothecium (Phillips, 1987), although germination at different temperatures varies between isolates collected from different geographic origins. This result, and the extensive distribution of S. sclerotiorum, suggests that this species is adapted to a much wider range of environmental conditions than S. minor (Young et al., 2004;Clarkson et al., 2017).
Since the northern piedmont region of Yin Mountain is the only region where S. minor was collected from diseased sunflower plants, this study was to determinel if low temperature could affect the biological characteristics of both S. sclerotiorum and S. minor isolated from this region and thus affect the isolate aggressiveness. Our results showed that not only mycelium growth rate increased, but also OA secretion ability and PG enzyme activity were boosted after culture at 4°C, suggesting that aggressiveness was also enhanced accordingly. Additionally, the average value of the key pathogenic factors of S. sclerotiorum such as OA value and PG enzyme activity was much higher than that of S. minor, indicating that genetic differences between S. sclerotiorum and S. minor may cause the response differently to temperature variations. The result also provided an exaplanation why S. sclerotiorum is the dominant causal agent for SWM worldwide, whereas, S. minor is only observed in certain sunflower planting regions which have typical environmental condition (Najafzadeh et al., 2018).
In this study we also showed that low temperature treatment can significantly enhance the aggressiveness of both S. sclerotiorum and S. minor, but increased value for S. sclerotiorum was greater than that of S. minor. This agrees with earlier observation that the pathogenicity of S. minor isolates collected from rapeseed in the central region of China showed mild pathogenicity compared to S. sclerotiorum (Yang et al., 2016). However, the molecular mechanism underlying such kind of phenomena is still poorly understood and needs in depth investigation in the future.