Streptomyces felleus YJ 1 : Potential Biocontrol Agents Against the Sclerotinia Stem Rot ( Sclerotinia sclerotiorum ) of Oilseed Rape

In order to explore the biocontrol potential of Streptomyces felleus YJ1 to sclerotinia stem rot of oilseed rape, we evaluated the effects of YJ1 as antagonistic strain on Sclerotinia sclerotiorum, including germination and formation of sclerotia, ascospore germination, mycelial growth and YJ1 colonization ability. We found the fermentation filtrate of YJ1 could inhibit sclerotia and ascospore germination and mycelial growth. In dual culture the inhibition zone diameter of YJ1 against S. sclerotiorum was 11.0 mm, and the inhibition rate reached to 80.26%. The ascospores germination was also significantly inhibited by YJ1 fermentation filtrate. In addition, YJ1 could colonize stably in rhizosphere and roots of rape. Otherwise, in the greenhouse we found the lesion would become smaller and slighter if the inoculated leaves were pretreated with YJ1 fermentation liquid. Therefore, our results strongly suggested that YJ1 was a promising biocontrol agent for control of oilseed rape sclerotinia stem rot.


Introduction
Sclerotinia stem rot of oilseed rape caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary is a widespread soil-borne plant disease (Yan et al., 2005).The fungus is capable of infecting a wide variety of plant species including soybean, bean, pea, lettuce, tomato, sunflower, rape and many other economically important crops (Hegedus & Rimmer, 2005;Fróes et al., 2012).Oilseed rape (Brassica napus L.) is the major oil crop in China.In recent years, S. sclerotiorum has an increasing threat to oilseed rape cultivation and become a potentially damaging disease.Sclerotinia stem rot can reduce the yield of oilseed rape from 15% to 60% (Subbarao, 1998).Due to its wide host-range, effective reproduction and sclerotia which can survive in soil for up to four years without hosts or favorable condition, it is very difficult to control S. sclerotiorum (Bae & Knudsen, 2007).
Many strategies were applied to control the sclerotinia stem rot such as crop rotation and fungicides (Mueller, et al., 2001).However, using crop rotation is unpractical due to the persistence of sclerotia in the soil for long periods (Demoz & Korsten, 2006).The use of fungicides has adverse effects on non-target organisms, and fungicides even lead to the selection of resistant populations (Huang, Bremer, Hynes, & Erickson, 2000).Therefore, biological control of sclerotinia diseases was interested as an acting substitute to other controlling methods.Biocontrol via antagonists such as Bacillus, Pseudomonas and mycoparasites Trichoderma spp.against the S. sclerotiorum was reported previously (Fernando et al., 2007;Abdullah, Ali, & Suleman, 2008).Some actinomycetes (mainly Streptomyces) have been also reported for the control of S. sclerotiorum, because these filamentous bacteria produce a wide spectrum of antibiotics as secondary metabolites and different types of enzymes, like chitins, cellulose, peptidase, glucose polymerase, glucanases etc. (Adams & Ayers, 1979).Many secondary metabolites or different enzymes exhibited strong antagonism against the fungal pathogens (Clardy, Fischback, & Walsh, 2006;Yuan, & Crawford, 1995).It exhibited a great application potential.
sclerotiorum and the colonization of YJ1 in the rhizosphere soil and roots of oilseed rape.This is helpful for us to have a fully understanding of the potential of YJ1 for biocontrol of sclerotinia stem rot of oilseed rape caused by S. sclerotiorum.

Strains, Culture Conditions
Streptomyces felleus YJ1 was maintained on Gause′s medium No.1.Gause′s liquid medium and millet liquid medium (including millet 10.0 g, glucose 10.0 g, peptone 3.0 g, NaCl 2.0 g, CaCO 3 2.0 g, distilled water 1000 mL, pH 7.2-7.4)were used for cultivating YJ1.Sclerotinia sclerotiorum was isolated from the sclerotia in the stem of rape from Wenjiang, China and maintained on Potato Dextrose Agar (PDA).YJ1 and S. sclerotiorum were stored at 4°C until required.

Preparation of Fermentation Filtrate
The strain YJ1 was cultivated for 4 days at 28°C on Gause′s medium No.1.The mycelium discs were put into sterile medium in 250 mL flasks.The flasks were incubated in the dark at 28°C on a rotary shaker at 160 r. min -1 for 7 days.The cultures (fermentation broth of YJ1) were centrifuged (8000 r. min -1 , 4°C) for 15 minutes to separate the supernatants.The supernatants were sterilized through 0.22mm bacterial filter and stored at 4°C until required.

Dual Culture Assay
The mycelial plugs (5 mm diameter) of YJ1 and S. sclerotiorum were placed on the same dish.The S. sclerotiorum were placed in the center of the dish and YJ1 were placed at 2.5 cm of the center inocula.The dishes were incubated at 28°C for 3 days.The inhibition zone was measured as the distance between YJ1 and S. sclerotiorum after 5 days.The experiments were repeated 3 times.

Antibiotic Activity Assay
Briefly, 1 mL sterile fermentation filtrate mixed with 9 mL melted PDA medium was replaced on a dish and 10 mL PDA medium was used as control.The experiments were repeated three times.And the dishes were incubated for 3 days at 28°C.Then, colony diameter was measured.The dishes were maintained at 28°C for a long time to observe the production of sclerotia.

Sclerotia Germination Test
10 sclerotia were selected to sterilize according to previous method.The sterile fermentation filtrate of YJ1 was used for soaking sclerotia for one hour, and sterile water was used as control.Next, the sclerotia were removed on PDA medium.After the dishes were incubated at 28°C for 2 days, we measured the germination of sclerotia every 24 hours.The experiments were repeated 3 times.20 sclerotia were selected to sterilize.The initial sterile fermentation filtrate of YJ1 at a ratio of 5 to 1 and 20 to 1 were used for soaking sclerotia for one hour, and sterile water was used as control.Next, these treatment sclerotia were buried in sterile sands.The favorable moisture was kept.Assessments were started the following spring when sclerotia germinate to form apothecia.

Ascospores Germination Test
The ascospores of S. sclerotiorum were collected to prepare the spore suspension (2×10 7 cfu/mL).The spore suspension was mixed with sterile fermentation filtrate of YJ1 to dilute 5 times and 20 times respectively.We also used sterile water as control.The mixtures were dropped on the glass slides and incubated at room temperature.The germination of ascospores was checked by using microscope after 24 hours.The experiments were repeated 3 times.

Streptomycin-Resistant marker Strains
The strain YJ1 was transferred to Gause′s medium No.1 which was added 20 µg/mL streptomycin, and gradually with increasing streptomycin concentration, such that at the maximum concentration used (500 µg/mL).Each time the concentration was increased by 20 µg/mL.Then the mutation frequency, the physiological characteristics of the mutant strain and its inhibition effects against S. sclerotiorum were checked.Finally, the mutant strains were incubated in the flasks that contained Gause liquid medium added with 500 μg/mL streptomycin.The flasks were www.ccsen put in the 2.5×10 7 cf

Dete
The oilsee inoculated of rape we made the d

Effects
When the control wa cm diamet repeated 3 3 Results We continued to detect the effect of YJ1 on S. sclerotiorum sclerotia formation.The results showed that the control began to produce sclerotia at five day, however, in the treatment group the sclerotia forming at least need seven days.Otherwise, we found in the Antibiotic Activity Assay the number of sclerotia were little than control at any different days.It indicated that the formation of sclerotia was greatly restrained by YJ1 (Table 1.).The inhibition rate of sclerotia formation was still 79.05% at 11 day.Thus, YJ1 could not only restrain the number of sclerotia production, but also delay the formation time.Note: The values in the table were the average of three replicates.Different letters in the same row represent significant differences (P<0.01).

Inhibition Effects of YJ1 on Sclerotia Germination
The sterile fermentation filtrate of YJ1 was used for soaking sclerotia.Then the sclerotia were put on the PDA medium.After 48, 72 and 120 hours, we found the sclerotia germination rates were always lower than control.However, after 168 hours all sclerotia germinated (Table 2).The results showed that sclerotia germination could be restrained by YJ1, but the inhibitory effects only maintained a short time.Eventually, the germination of sclerotia was not affected by YJ1 fermentation filtrate.
Otherwise, the sclerotia germination and apothecium production could be affected by different concentration of YJ1 fermentation filtrate.As seen in Table 3, the initial fermentation filtrate had extreme effects.The sclerotia germination rate was about 10%, while the control was 100%.The inhibition rate of apothecium formation was the highest in the initial fermentation filtrate.But with the filtrate concentration reducing, the inhibition rate decreased obviously.We also fo After appl different d quantity of the quantit increasing different in

Effects
The leaves YJ1 had an leaves (Fig

Discussion
In this study, we found that the mycelial growth of S. sclerotiorum and formation of sclerotia were inhibited extremely by Streptomyces felleus YJ1.Sclerotia germination was also restricted.The results were consistent with the reports by Zhu, et al. (2008) and Han, et al. (2011).In addition, apothecia formation and the germination of ascospores were also restricted obviously by YJ1.Sclerotium is an important survival structure, which can survive in the summer and winter or other extreme circumstances (Huang & Kozub, 1989).In favorable condition, sclerotium can germinate to form hyphae directly and also can produce apothecia to release a lot of ascospores (Abawi & Grogan, 1979).Therefore, sclerotium becomes a very important part in the life cycle of S. sclerotiorum.According to this, delay or inhibition of sclerotia germination can reduce the sources of primary infection, and the speed of disease epidemics will also be retarded partly.If ascospores germination was restricted, the sources of primary would be reduced largely.biocontrol of strain YJ1 against S. sclerotiorum exhibited a potential application.
Using antagonistic microorganisms to control plant diseases, the key is to determine whether the strains can colonize in the target crops or their surrounding environment and whether the strains can act antagonistic function or not (Lian et al., 2011;Chen et al., 2011;Zhu et al., 2005).It is necessary to make sure the colonization ability of the strains as well as their antagonistic effects after colonization (Guo, 1998).Many scholars such as Yuan & Zhou (2006) and Guo, et al. (1996) considered these as important indicators to evaluate the antagonistic strains.In this study, the strain YJ1 could colonize stably in rhizosphere and roots of rape.The quantity was large and the strain could frequently keep for a long period.The similar results were obtained by Guo (2005), and Wang, et al. (2002).Our results indicated that Streptomyces felleus YJ1 had a good prospect as a biological control strain.
In summary, Streptomyces felleus YJ1 had strong antagonism against Sclerotinia sclerotiorum and good potential for colonization.It is apparent that Streptomyces felleus YJ1 has great potential to control oilseed rape sclerotinia stem rot caused by S. sclerotiorum.
Figure 3 E l; B: Treated b

Figure
Figure

Table 1 .
Inhibition effects of YJ1 on the quantity of sclerotium production

Table 2 .
Effects of YJ1 on sclerotia germination