The Viability Detection Of Fusarium Spp.

Fusarium was a kind of fungi with great morphological variation and with high economic significance. So many attentions were paid on Fusarium classification and great efforts were made.Fusarium could be identified with morphological methods,immunological methods and molecular methods.But the sample pretreatment of morphological methods was complicated and time consumed,and there were cross-reactions, high false positive and false negative rate for immunological and molecular methods.Matrix-assisted laser desorption-ionization time of flight mass spectrometry(MALDI TOF MS) was an analytical chemistry technology,it could provide mass spectra containing different biomarkers specific to various microorganism. Based on the spectra with the microorganism specific biomarkers,the microorganism can be detected and identified.MALDI TOF MS was applied to microbiological assay and classification has some advantages compared with conventional methods:simple and rapid,good reproducibility,perfect accuracy,well automation,high-throughput,and so on.35 strains of Fusarium originated from CGMCC,USA and Argentina were used for MALDI TOF MS anlysis.In this study,Fusarium proliferatum var.minus and Fusarium graminearum were analyzed by MALDI TOF MS used for setting up a general sample preparation protocol.The present study mainly employs two different sample preparation protocols:treating fungi by solvent(single treatment and combined treatment) and enzyme(snail enzyme).At the same time,different experimental factors such as solvent system used,matrix,solvent of matrix,fungi quantities,sample application method,sample application quantities,times of washing,ultrasonic treatment time were investigated.The role of different factors in sample preparation was systematically illuminated.The optimum experimemtal condition for analysis of Fusarium by MALDI TOF MS was got for the first time:the optimum solvent for mycelium treatment was 70%formic acid:acetonitrile.The optimum matrix and solvent of matrix were HCCA and 50ACN:TFA:water(20:1:19).The optimum amount of mycelium was about 8mg.The optimum sample application was improved dried droplet method.The optimum sample application quantity was 1μl.The optimum times of washing were once.The optimum ultrasonic treatment time was 10 min.This protocol could be used to differentiate the fungi in different species of Fusarium and different strains of Fusarium virguliforme according to the specific peak profiles in mass range 2000-19970Da.And the reproducibility was proved perfect using this protocol.This study laid a foundation for establishing the standard mass spectra database of Fusarium.A protocol of identification for Fusarium by MALDI TOF MS was firstly established:fungal mycelium(about 8mg) was washed once with deionized water,and formic acid, acetonitrile were followed,and the pellet was treated with ultrasonic for 10min,then the pellet was centrifuged and supematant was applied to the MSP plate using improved dried droplet method.Matrix HCCA was dissolved into CAN:TFA:water(20:1:19).Treated in water bath under 5 temperature gradient from 43℃to 47℃for 1min to 5min,the viability of F.virguliforme,an exotic quarantine pathogen originated from USA and Argentina,was analyzed using Guava Easycyte Mini after fluorescence staining.The traditional spore germination test was set up as the control.The results showed that the pathogen was unvital treated with 47℃for 4min.Statistical analysis using SPSS software indicated that very significant negative correlation was found between the pathogen viability and temperature.Similar result was also obtained between the viability and duration of treatment, except for strain 2-1 and 22825 significant negative correlation between viability and duration of treatment. Between spore germination rate and temperature as well as duration of treatment assumed very significant negative correlation,whereas between the fungus viability and the spore germination rate had very significant positive correlation.Both the protocol and the mass spectra database of Fusarium were firstly established.And the mass spectra database of Fusarium included 35 strains of Fusarium.So a new way was opened up for rapid, high-throughput,automatic detection of fungi,and a new detecting platform was put up.And the viability of F.virguliforme was analyzed using Guava Easycyte Mini after fluorescence staining;it was proved that the procedure was completed for the first time.This study revealed that the traditional spore germination test could be replaced by the pathogen viability detection procedure,and the detection duration was greatly shortened from 24h to 20min and the detection efficieney was improved obviously.This procedure was of important reference value for the establishment of viability detecting platform of quarantine plant pathogenic fungi.