Studies On Microbiol Diversity In Sclerotinia Sclerotiorum Sclerosphere And Screening Of Antagonistic Bacteria

Rapeseed is widely cultivated in China and an important oil crops. Rape sclerotinia disease is the major disease in rape production in China, especially in the middle and lower reaches of the Yangtze River region, affecting the rapeseed yield and quality improvement. As rape sclerotinia disease pathogen, Sclerotinia sclerotiorum, in the field mainly exists in the form of sclerotium, infection period will scatter ascospore currents transmission, prevention and control of rape Sclerotinia obviously has great difficulty. Due to lack of high variety, pesticide use also has many problems. Biological control has gradually become a research hotspot. Because of host specialization, good prevention effect and other reasons, Coniothyrium minitans attract attention of many scholars.Due to Sclerotinia sclerotiorum survives as sclerotia in soil most of its life cycle time, it has been proved that soil microbes is the main factors influencing the sclerotia survive; And after applying Coniothyrium minitans also survives in the soil in spore form. The relationship between soil microbes and Coniothyrium minitans directly affect the efficiency of Coniothyrium minitans parasitic sclerotium. Thus study sclerotium surrounding soil microbes for prevention and control of rape sclerotinia has great theoretical value and practical significance.Defined distance around3mm of sclerotium epidermis of soil for sclerosphere soil, while the soil far away from the sclerotia (over5cm) is defined as non-sclerosphere soil. Comparison the difference of microbial species and amount of the two, to determine weather the sclerosphere effect exists or not. Separate and identify the culturable microorganisms in soil around sclerotia, and study the relationship between these microoganisms and Sclerotinia sclerotiorum, also the interactions with Coniothyrium minitans, looking for the new way to enhance and stabilize the effect of Coniothyrium minitans mildew control.Using T-RFLP fingerprint technology detect sclerotium scarf with the bacteria and fungi in the soil from sclerosphere. Through cluster and diversity analysis, we have found that for fungi, the number13sample were classified to a single branch because of its high diversity index (D=0.91, E=0.92) compared with other samples; for bacteria, the number3sample also has been classified to a single branch, because of the same reason (D=0.94, E=0.82), the number10,19,22samples have a very low diversity index (D<0.67, E<0.61) witch caused a detectable difference with other samples. The change of fungi species have no significant relationship with neither the depth、time nor site of sampling (P>0.05), but for bacteria, the change were related to sampling depth (P=0.002), y=11.208+3x.Through a period of1year of continuous sampling, we found that at all depth, the change of bacteria content in sclerosphere soil is not completely consistent with non-sclerosphere, but in the late sample, both trends and bacteria content uniformity. Sclerotium bacteria content of soil around the depth change also is not the same, surface soil bacteria content variation is not obvious, sclerotium around5cm and10cm depth of soil bacteria content change relatively consistent. Definition the ratio of sclerosphere with the non-sclerosphere confining effect of soil microorganism quantity for sclerosphere effect value, microorganism content in soil from sclerosphere is higher than non-sclerosphere soil (W>1), witch was found by calculate sclerosphere effect value.Of sclerotia in soil around separate identification of culturable bacteria and fungi, total have21species of bacteria and12species of fungi. For bacteria,8species belong to Pseudomonas,6species are Bacillus and3section of Arthrobacter, the remained four species are Brevibacterium frigoritolerans, Chryseobacterium formosense, Klebsiella, and Burkholderia. For fungi,5species belong to Penicillium,2species of Neurospora, the remained five are Rhizopus oryzae, Hypocrea pachybasioides, Bionectria ochroleuca, Aspergillus niger and Lasiodiplodia parva respectively.21representative strains of the appraisal bacteria were chosen to analysis the antagonism relationship with Sclerotinia sclerotiorum and with Coniothyrium minitans. By PDA plate confrontation training, a total of17strains of nuclear dish bacteria show antagonism effect of bacteria, of which4strains have remarkable antagonism effect, among them, a-B2and a-B9belong to Bacillus, B4belongs to Pseudomonas and B11belongs to Arthrobacter. For Coniothyrium minitans,15strains have inhibition effect on the growth of Coniothyrium minitans hypha, most of which show a strongly inhibition effect; We also screening to6strains were found to have few antagonist effect on Coniothyrium minitans, respectively, belong to Bacillus (B9), Pseudomonas (B15, a-B20, d-B7) and Arthrobacter (B16, a-B7). By contrast, we found that the Sclerotinia sclerotiorum antagonist effect of bacteria also can significantly inhibit the growth of Coniothyrium minitans, meanwhile, bacteria which show no inhibition to Coniothyrium minitans also have poorly antagonism effect on Sclerotinia sclerotiorum, and the effect is unstable.