Study On The Activity Detection Of Monilinia Fructicola(Winter) Honey Living Spores

Monilinia fructicola is an important worldwide pathogen,causing billions of economic losses and widely spreading in Europe,Asia,and South America.In our country,both kernel fruits and stone fruits are infected,such as apple,pear,peach,plum,etc.Moreover,the climate in our country is suitable for mycelial growth and survival once it is transported in.The pathogen was easily hided in fruits and spread through trade,for why Monilinia fructicola was more than once detected in inbound fruits in recent years.Monilinia fructicola was list in quarantine objective in our country owing to easily spreading and potentially threatening.In the world,most research on Monilinia fructicola were focus on molecular biology,morphological identification and drug resistance testing,lacking of the rapid detection of activity of the pathogen conidium.Therefore,it is imperative to establish a rapid,accurate,reliable,and referential method for quarantine fungito detect the conidium activity.Based on this,we studied on detecting the activity of Monilinia fructicola conidium,and had some results below:1.The spore suspension,made from the conidia Monilinia fructicola produced on PDA,was taken a portion of 100? water bath for 15 min to inactivate.Inactivated and live spores were fluorescently stained with 10 kinds of reactive dyes.Stains were selected based on obvious differences in dyeing between the two states spores by fluorescence microscope.Among them,FDA and PI are the best dyes.2.The optimum staining conditions of FDA and PI were optimized by comparing the results under 5 different concerntrations and durations respectively.PI has an optimum staining condition with a concertration of 0.05 mg/m L and lucifugal for 4 min,while FDA with a concerntration of 0.5 mg/m L and lucifugal for 16 min.3.Both of two states of conidia were treated with different combinations of 5 temperatures and 5 times respectively under the best staining conditions of FDA and PI.The results showed that the spores staining rate of different treatments were not consistent with the spore germination rate.Under some treatment conditions(50?-20 min,55?-10 min),spore germination test results showed that all spores died,but spore staining results under these conditions showed that some spores were still active(FDA staining rates 84.89% and 91.44% for the these two conditions,respectively).it could be implied that the standard of spore staining activity was more accuratly than spore germination.The germination rate is much lower than the spores staining rate which is very likely that the spores are inactivated by the tested temperature stress and cannot be germinated.4.In view of the feature of colorating activated spores for FDA and colorating inactivated spores for PI,a higher accuracy of activity detection was guaranteed by verifying each other.Based on this,a co-stainingsystem of PI-FDA was set up to detect two strains with different biological characteristics.In this study,a detection technology system of activity of M.fructicola was set up,based on PI-FDA co-staining and laser confocal scanning microscope.Through this method,not only the status of conidium is described more accurately,but also the time-cost of detection is reduced from 2 or 3 h,the time of spore germination,to 1 h.It could be used to quarantine M.fructicola at the port,and be a reference for other plant pathogenic fungi.