| The sclerotia of Sclerotinia sclerotiorum germinate in a suitable environment to produce apothecia and eject outward ascospores for infection and spread of the disease caused by the fungus.Getting enough sclerotinia ascospore is an extremely important technical aspect in studying the pathogenesis of S.sclerotiorum and screening of antimicrobial rape variety,but acquiring the ascospores is more time-consuming in natural conditions,therefore it is necessary to study sclerotium indoor germination and ascospore induction.As we were all known,there are variety of methods to induce ascospore formtion indoor,but the results of these experiments and the expected results make a greater difference,with a low germination rate,and producing little apothecia.In addition,some of the experimental methods are relatively time-consuming to implement it.In this paper,by comparing previous studies,the cultivation of sclerotia,germination of sclerotia,and the induction and collection of ascospores were investigated in order to explore and establish a set of suitale experimental method for the production of sexual spores of S.sclerotiorum.The main results are as follows.1.Effects of different media on the size,quality,quantity,and subsequent germination of sclerotia of Sclerotinia sclerotiorumIn this experiment,wheat,potato,sunflower faceplate and PDA were selected as medium to culture S.sclerotiorum,in order to get a lot of artificially cultivated sclerotia of the fungus.The results showed that on potato medium,the produced sclerotia are mostly irregular or long elliptic,the average size of single grain sclerotia could reach 1.02×0.39 cm,and they have good texture and quantity(in each triangular flsk about 600 grains of sclerotia could be produced).However,only the sclerotia cultivated on potato medium could well produce apothecia.On PDA medium,the developed sclerotia were low in production(around 37 grains of sclerotia per dish),mostly were round or oblate,immature in the majority,easy to fracture,and also the smallest(0.45×0.16 cm)on average.In the subsequent germination experiment,the sclerotia only grow hyphae,but could not produce ascogonidium The sclerotia produced on wheat medium were between the above two types.They were mostly round,grow mature in the majority,with the average size of 0.72 cm × 0.28 cm.They could only germinate ascogonidium,but did not produce apothecia(invalidgermination).On sunflower faceplate,it was difficult to inoculate the fungus,with easy infection of bacterium and no sclerotium produced.2.Influences of different moisture-keeping materials on the germination rates of sclerotia induced at low temperature of 4 ℃This experiment indicated that the experimental strains without induced germination at 4 ℃ low temperature and moisture keeping could not produce ascogonidium,only growing hyphae.The five kinds of materials with moisturizing function,vermiculite,quartz sand,perlite,sterilized soil and absorbent cotton,were applied to germination induction of the sclerotia under cold and wet condition for 2 months at the temperature of 4℃.The results showed that vermiculite and pearlite had general moisturizing effect,the sclerotia tested easily grow hyphae,but didn’t produce ascus handle during germination induction.Use of sterilized soil for sclerotium moisturizing at low temperature induction period,because of easy breeding of bacteria,caused the sclerotia rotting,and no sprouting sclerotium ascus handle,with a large number of hyphal formation.In quartz sand after adding water,because of the low permeability,only a fraction of sclerotia germinated to produce ascus handle,with the germination rate of about 53%.In absorbent cotton,with good water keeping,easy to sterilization,and good permeability,the tested sclerotia easily germinated to produce ascus handle,with a high germination rate of 95% during the period of low temperature induction.The results also idicated that the induced germination during the period was invalid,producing only ascus handle but no apothecia,In addition,the sclerotia which did not produce ascus handle during the period of low temperature induction subsequently did not germinate to form apothecia under the 18 ℃ normal temperature induction.3.Influences of different moisture-keeping materials on the germination rates of sclerotia induced at normal temperature of 18 ℃In this experiment,absorbent cotton,vermiculite,perlite,sterilization and filter paper were selected as moisturizing materials to induce the germination of the sclerotia of S.sclerotiorum after the 5 ℃ low temperature induction.All treatments were under the condition of 18℃ normal temperature and illumination of slightly greater than 500 Lux.The results showed that the three moisturizing materials of filter paper,vermiculite and pearlite had a similar sclerotium germination situation,in which the tested sclerotia formed only the handles,gradually wither after 3 to 5 days,and couldn’t produce apothecia.The sclerotia moisturized with sterilized soil werecontaminated most by bacteria during induction of germination to rotting,with a large number of hypha but no asaophores.As well as low temperature induced,absorbent cotton moisturizing treatment was the best among the five moisturizing materials,with the average effective germination rate of 59%.4.Comparison of different collecting methods in the number and aridity of ascospores of S.sclerotiorumThe three methods,ascospore natural ejection,washing and vacuum suction,were adopted to collect ascospore of S.Scleotiorum in this experiment.The results showed that all of the three collecting methods could collected ascospore with activity.Using water washing method,the average number of spores collected per unit area(0.01 mm2)was the most(21),but the collected ascospore germinated easily in water,not convenient to save.Using vacuum suction method,the average number of spores collected per unit area(0.01 mm2)was the next(12),and the spores were relatively dry,easy to save.The number of ascospores collected per unit area by use of natural ejection method was about 2,and next to vacuum extraction method in ascospore aridity.5.Establishing of the induction method of apothecia and ascospores of S.sclerotiorumBased on the results mentioned above,a set of practical and effective induction methods of apothecia and ascospores of S.Sclerotiorum was etablished as follows:Potato medium was used to cultivate a large number of sclerotia at 20 ℃ in illumination,and absorbent cotton was choosed as the moisturizing material in low temperature induction at 4 ℃ in dark.After two months of low temperature induction,the tested sclerotia were moisturized with absorbent cotton at 18 ℃ and in illumination of slightly greater than 500 Lux to induce germination.Finally,the vacuum suction method was taken to collect the ascospores.The set of methods was proven to be practical and effective for the induction of apothecia and ascospores of S.Sclerotiorum... |
PDF Full Text Request