Studies On Screeninq Identification And Application Of Beneficial Microorganisms Isolated From Aging Tobacco Surfaces

Biodiversity is the survival and developing basic for human society. It has been regardedas research focus to explore the microorganism of special and extreme environment recently.As the biggest tobacco planting and spending country in the world, our researchers should paymore attention to the study of microbial diversity of aging tobacco surface which is almost thevirgin land in China. Hence, we have studied the microbial flora composition, bio-controlstrains and TSNA degrading strains of aging tobacco surface from staple tobacco region ofour country in depth and systematacially.1618microbes, including1279bacterium,226actinomycetes,75fungus and38yeast,have been isolated from14species aging tobacco surface using the conventional methods.The species and numbers of microbe isolating from aging tobacco surface reduced evidentlywith the extension of aging period. Microbial species and numbers of high quality tobaccowas more than inferior tobacco. The microbial species and numbers under different levels,aging periods and fermentation conditions showed dramatic difference.BC79isolate, displayed strong inhibition against5common tobacco pathogenic fungi,was screened from the microbial library of aging tobacco surface. Based on morphological,biochemical, physiological and chemotaxonomic analyses as well as phylogenetic16S rDNAsequencing data, BC79was identified as Bacillus methylotrophicus and genbank accessionnumber was JF449444. In this study, B. methylotrophicus BC79isolate was reported asbio-control microbe for the first time. This strain was able to suppress mycelial growth andconidial germination of tobacco grey mould in dual cultures on solid media, which infectsome bioactive ingredients had been secreted by BC79. We therefore assessed fermentationconditions and stress resisitance for the study of B. methylotrophicus BC79to explore thebiggest antibacterial ability. Carbon and nitrogen are two important elements for organisms tolive and grow. We observed pronounced differences in antifungal activity to B. crease usingeight different carbon and nitrogen sources. Sucrose and starch were better carbon sourcesfor the BC79strain, and were superior to the control (glucose), other carbon sources testedshowed less biological activity compared with glucose. Sucrose was the optimal carbonstudied. As a universal nitrogen source, peptone was proved to be the best option for strain BC79. TSA was superior to PDA isolation medium, and suited for a BC79response to B.crease. This isolate entered exponential phase at10h, and terminated at18h. Careful studyof additional fermentation condition showed that filtrates had highest antifungal activity whenBC79was cultured at pH7.0,170r/min,30oC for48h. We found no obvious difference afterseven transfers. Inhibition diameter fluctuated only slightly, indicating that the antibioticability of BC79declined only moderately and tended to stabilize. Exploration of fermentationconditions of BC79conducives to the maximum stability of bio-control activity. The B.methylotrophicus BC79strain colonized tobacco plants quickly. BC79fermented liquidshowed better control effect to tobacco grey mould in green house and growth-promoting totobacco seedlings, suggesting that the BC79isolate might be exploited as a promisingantifungal agent against B. ciease.We screened bacterial strain that efficiently transform nitrite and nitrate from microbiallibrary by enrichment and domestication screening methods.2culture mediums combinations,SM+DM1、DM2+BTB, could be regarded as the optimal combinations for isolating goalmicroorganisms. Based on morphological, biochemical, physiological and chemotaxonomicanalyses as well as phylogenetic16S rDNA sequencing data, AS97was identified asPseudomonas fluorescens with Genbank accession number: JF449445. This research showedthat BTB medium was an effective and reliable way for isolating and screening bacteria onthe basis of colour change of culture medium. Additionally,5%inoculation of AS97isolatesuspension was optimal compared with other two treatments, and the optimal fermentationtemperature was30℃with60%relative humidity. AS97isolate had strong capability todegrade nitrite, nitrate and TSNA. Correlational analyses showed that there was significantrelationship between the content of those three substances presented, from which we couldinfer that nitrite and nitrate were the precursors of TSNA. We know that AS97isolatefermentation culture also displayed obvious promoting affect for tobacco quality from smokepanel test, which indicating AS97isolate has drastic practice value and potential development.AS97isolate also showed better resistance and had wide growth temperature and pH value. Inour study, the optical growth temperature and pH have been explored, which laid solidfoundation for the deep application of strain AS97for degrading TSNA in the productionpractice.