Identification Of Antagonistic Fungus Strain PY-1 And Evaluation Of Suppression Of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is a plant pathogenic fungus found inhibiting soil ubiquitously in many parts of the world. In China, this pathogen causes stem rot on oilseed rape (Brassica napus), and causes serious losses every year. Biological control is a useful alternative to chemical fungicides against Scleriotinia diseases. In this paper, a antagonistic fungus strain PY-1 which showed strong inhibition against S. sclerotiorum was screened out, and the taxonomy of PY-1 strain was identified. The potential for biological control of oilseed rape stem rot was evaluated, and the cultural conditions for the fermentation of antifungal substance (AFS) was optimized. The stability of AFS was examined under several conditions, the AFS also was extracted from fermentation liquid and partially separated and purified by HPLC, and two active components were primary identified. The main results were listed below.1. Identification of PY-1 strain: PY-1 strain was identified as Penicllium oxalicum based on the characteristics of the colony morphology, conidiophore, phialides and conidia, and the identification was confirmed with alignment of ITS sequence of PY-1 and that of other fungi on GneBank.2. The mechanism of antagonism on S. sclerotiorum by strain PY-1: Strain PY-1 showed strong inhibition to the mycelial growth of S. sclerotiorum on PDA plates. The inhibition zone could remain for more than two months. One milliliter of fermentation liquid in 19 ml PDA could inhibit the mycelial growth of 5. sclerotiorum significantly, the inhibition effect was 62.24 %. Comparing with Carbendazim, the relative activity of anti-fungal substance of PY-1 produced at the third day was about 4μg a.i. per millilitre. Strain PY-1 could neither parasitize the sclerotia of 5. sclerotiorum, and nor suppress the mycelial germination of the sclerotia, but it may slightly reduce the product of sclerotia in PDA plate。3. Biological control potential of oilseed rape stem rot: (1) Both spores and fermentation liquid of PY-1 could suppress the infection of S. sclerotiorum on detached oilseed rape leaves. With an application of 10⁷ spores/ml on leaves, average diameter of lesion being 0.73±0.64 cm, the size of lesions were significantly reduced comparing to water-treated control with average size of 4.07±0.68cm, and only 75% tested leaves could be successfully induced to form lesions. Only one lesion on detached leaves was induced among 15 treated leaves with the undiluted fermentation liquid, and the lesion was 1.0 cm. (2) Both spores and fermentation liquid of PY-1 could suppress the infection of S. sclerotiorum on living oilseed rape plants. With an application of 107 spores/ml on plants, the average diameter of lesion was 0.73±0.64 cm, and only two leaves produced lesions in all of treated plants, while more leaves were infected and the average size of lesion was 2.80±0.5 cm in water-treated control. Only one leaf was induced to produce a lesion among all fermentation liquid-treated plants. (3) Field experiments in 2007 showed that strain PY-1 (including spores with a concentration of 1×10⁷ spore/ml and 5 fold diluted fermentation liquid) could significantly suppress sclerotinia stem rot of rape seed, the relative biocontrol efficiency were 52.65 % and 39.48 % respectively, while the effect of dimathchlon was 31.04 %.4. Induced resistance of strain PY-1: Acquired resistance of cotton against Colletotrichum gossyoii was observed when treated cotton roots with spores (10⁷spores /ml) and fermentation liquid of strain PY-1. If cotton plants were treated with spores 15 d ahead of inoculation of Colletotrichum gossyoii, the disease index was suppressed with a rate of 66.92%, however, only 34.21% was achieved with fermentation liquid.5. Facts affecting the production of AFS of Strain PY-1: (1) Production of AFS of strain PY-1 was related with the inoculation quantity, fermentation temperature and primary pH value of medium. The optimum fermentation conditions for inoculation quantity for 0.2-0.4 ml of 1.5×10⁷ spores/ml), and primary pH of medium ranged from 4 to 5, and fermentation temperature for 20℃were benefit for the high production of AFS. Furthermore, packaging volume of medium in flask which may influence the aerial condition did not effect the production of AFS significantly. (2) Effect of carbon sources and nitrogen sources on AFS production was studied using CMS medium as basic media. In the carbon source experiment, four (sucrose, glycerol, trehalose, mannitol) out of nine carbon source could be the most optimum by PY-1 for AFS production, and three nitrogen source including L-Arg,fish powder and peanut powder were the most optimum nitrogen source. PDB medium was suit for AFS production also. (3) The integrative condition of optimum medium and fermentation conditions for production of AFS of PY-1 strain were studied, and results showed that the optimal fermentation medium was KH₂PO₄,1.0g; MgSO₄.7H₂O,0.5g; CuSO₄.5H₂O,0.005g; KCL, 0.5g; L-Arg, 0.871g ; sugar, 10.12g. The pH value, reducing sugar and total sugar, ammoniacal nitrogen and biomass were changed during process of fermentation, and AFS production could y reach a peak on the 3rd day. 6. The antagonism range of strain PY-1: Strain PY-1 showed significant suppressing the growth of many fungal pathogens, such as Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporhides, Gibberella zeae, Magnorpthe grisea, Monilinia laxa, and Verticillium dahilae. However, strain PY-1 did not show any effect the growth of Rhizoctonia solani and Sclerotium spp, and plant pathogenic bacteria.7. Some primary properties of AFS produced by strain PY-1: The extract from fermentation liquid with aether showed significant anti-fungal activity against S. clerotiorum, and was used as crude AFS for testing the stability under given conditions. The AFS was very stable to high temperature when treated with at 121℃for 30min, it was also acidic or alkaline-stable, the activity of AFS was not significantly reduced when treated in buffers with a wide range of pH values (3-12); and interestingly, the antagonistic activity was improved when treated with UV-irradiation.8. Primary identification of the AFS produced by strain PY-1: Paper chromatogram analysis indicated that the AFS produced by strain PY-1 was a fat-soluble and ambisextrous antibiotic.9. Primary separation of the AFS produced by strain PY-1: The AFS existed both in mycelial and fermentation liquid of strain PY-1 and could be extracted with aether and chloroform, suggesting that the AFS was a low polarity substance. The optium extract proportion for the extraction of AFS was 3/4 volume of ethyl acetate.10.Primary purification of the AFS produced by strain PY-1: (1) Three substances were separated from the AFS in solvent system (Ethyl acetate: methanol: H₂O=100:10:l) though TLC, and the substance with a Rf of 0.89 showed strong antagonistic activity. (2) The activity band on the thin lay were collected as crude sample of the AFS, and further separated with HPLC, and a second round separation with HPLC was carried out to obtain two active compounds a1 and a2 using a mobile phase of 70% methanol,30% double distilled H₂O. The retention time of al was 11.038 min, and that of a2 was 13.598 min with a flow rate of 0.8 ml min-1. (3) The molecular weights of a₁ and a₂ were examined with EI-MS, and the results showed that the molecular weight of a₁ and a₂ were 274.2 D and 326.1 D respectively.