? Morphology And Development Comparision,and Ultrastructural Study Of The Influence Of Light On Two Groups Of Botrytis Cinerea ? Disease Detection And Pathogens Identification Of The Black Rot Tissues In Persimmons

Botrytis cinerea Pers.:Fr.is a complex and model species of the genus Botrytis.It could infect a variety of hosts and cause gray mould disease on more than 200 crop species.Being able to infect at low temperature,it is a serious threat to a variety of harvested fruits and vegetables,such as grapes,strawberries,and tomatoes.In previous study,two kinds of pathogenic symptoms of the gray mould were found on diseased grapes during storage at low temperature.Through the analysis of the growth and virulence phenotype on host and in vitro,a new group of B.cinerea was found to be significantly different from the classical taxonomic model strain in sporulation characteristics.As this group is very harmful with the characteristics of fast and abundant sporulation,further study of the morphological characteristics and development will provide useful informationfor the classification of subspecies of B.cinerea.In this study,strains GBot-0023,the typical representative of new taxon of B.cinerea,and the model strain B05.10 were used as experimental materials.The morphological differences and development processes were compared between GBot-0023 and BOS.10 by using optical microscope and electron microscope.The reasons of different ability in sporulation between these two taxa were also analyzed.Moreover,transmission electron microscopy was used to compare the varied influences of light qualities on the hyphae and spores between the two taxa.Results obtained are as follows:1.Scanning electron microscope observation showed that compared to the model strain B05.10,the new group GBot-0023 had obviously different sporulation patterns.B05.10 could produce a lot of microconidia.The macroconidia of B05.10 was bigger and sculpture was shallow,while GBot-0023 mainly produced smaller macroconidia with deeper sculpture.Transmission electron microscope observation showed no obvious difference in the ultrastructure of hyphae between GBot-0023 and B05.10,but significant difference in the ultrastructure of spores.Most of volume was occupied by storage body and little mitochondria and other organelles can be seen in the cytoplasm of BOS.10 spore,while there were small storage body and large number of mitochondria in the spore of GBot-0023.The big and stable differences in sculpture and ultrastructure of conidia make them useful references for dividing gray mould pothagens.2.GBot-0023 has the same development process as B05.10 except the timing of morphogenesis of certain structures.The formation of heptera in B05.10 was slower than that in GBot-0023,which may be one of the reasons for the dissimilarity of the sporulation rate between these two strains.Further study found that low glucose(0.5%)could accelerate the development of heptera in B05.10,thereby accelerated the sporulation of B05.10.High glucose(5%)showed certain inhibition on the process of sporulation from conidiophore.The results indicate that the time for differentiation of heptera is probably the key point for the difference between the two groups in sporulation.3.Compared with B05.10,GBot-0023 can sporulate rapidly and numerously in low temperature without providing nutrients,and has strong adaptability to the changes of nutrients and temperature.When GBot-0023 was cultured on PDA medium at 25?,the hypha were atrovirens,the colony grew without any obvious circadian patterns,and spores were produced numerously after 3days incubation.In contrast,the hypha of B05.10 was grayish and fluffy,colony growth appeared to be with circadian rhythm,and sporulation lags obviously.When cultured on different nutritional mediums,such as glucose,tomato and grape juice and MY,the colony morphology of B05.10 changed significantly and certain special nutritional conditions was capable of promoting the sporulation of B05.10;but there was no significant difference between the growth and morphology of the colony of GBot-0023,After 45 days cultured at 4?,B05.10 could form a large number of sclerotia on the surface of colony and no conidia was produced,while GBot-0023 sporulated normally but didn't produce sclerotia after 3 months incubation at low temperatures.GBot-0023 and B05.10 have the same response to light.Blue light can inhibit their sporulation and pigment formation.4.Different lights and illumination time have different effects on the hyphae of GBot-0023 and B05.10.Under the treatment of blue light for 24h,mitochondrion swelled accompanied by cristae vacuolar distortion.With the prolongation of the time of illumination(72h),mitochondrial distortion was recovered.The red,green and blue light could induce concentric vortex shape deformationin the mitochondria of GBot-0023 hypha,however,prolongation of time of illumination(72h)couldn't recover the mitochondrion.Different light quality had no obvious effect on ultrastructure of GBot-0023 and B05.10 spores.To sum up,the two groups of Botrytis cinerea have great difference in colony morphology,spore morphology,sporulation,growth process,utilization of nutrients and response to temperature.The time for forming heptera could be the key point for the difference in sporulation of the two group.Representative strain GBot-0023 of new taxon has strong adaptability to environment,which need more concern and attention.Blue light treatment could induce hyphal mitochondrial damage of GBot-0023 and B05.10,and inhibit their sporulation,suggesting a potential physical measure to control B.cinerea.Pathogen infection is the main reason to cause decay on postharvest fruits and vegetables,which also brings the huge losses to our country's economy.Persimmon is one of the main fruits of the fruit market in Shanghai,and because of its sweet taste,rich nutrition,persimmon is favored by the majority of consumers.For nearly three years of research,we found,however,some persimmons with healthy appearance often contain a large number of granular or massive black materials inside.Due to such symptoms occurs under the peel,it is hard for the consumers to distinguish the bad fruit only from its appearance.It seriously affects the food safety and shelf life of persimmons.Predecessors' research mainly focus on research of persimmon disease in cultivation period before harvest,but rarely on market diseases.Pathogens detection and identification of the black rot tissues was made based on investigation of retail and wholesale market sales situation to explore the cause of formation,and in order to ensure the safety of the persimmon supply.The results are as follows:1.Persimmons in Shanghai fruits markets mainly come from Guilin,Dongtai,Guangdong,Shanxi and Zhejiang,Hangzhou and other places.Among these import places,Guilin is the major source.The disease rate of persimmons sold in Shanghai is as high as 85.52%.The disease can be divided into two types according to the symptoms:"granule black rot" and "fruit top black rot".Pulp granule black rot accounted for 60.94%of the total number of diseased persimmons,while fruit black rot accounted for 24.58%.During the period of sampling surveys,persimmons from Guilin,Shanxi and Guangdong mainly had the disease of pulp granule black rot Morbidity of persimmons from Guilin and Shanxi was 100%,while that of Guangdong was 87.5%.Persimmons from Dongtai and Hangzhou mainly had the disease of fruit top black rot.Morbidity of persimmons from Dongtai was 16.67%in 2010,and 80%in 2011,with significantly increasing trend;Morbidity of persimmons from Hangzhou was 100%2.The suberization in the black rot granules,mycelium in the black rot tissues at the top of the flesh and in some black rot granules could be observed by scanning electron microscope.Pathogen separation of the black rot particles from persimmons in different regions for three years shows that the carrier rate is 29.82%on average.The detection rate of black rot granules from Guilin in 2010,2011 and 2012 were 66.67%,8.89%,9.50%.Pathogens are always detected from black rot tissues at top of the persimmons.A variety of pathogens were detected from the disease tissues,including Cladosporium spp.,Alternaria spp.,Penicillium spp.,Fusariumspp.,Colletotrichum spp.,Pestalotiopsis sp.,Botrytis sp.and many other fungi and bacteria.Cladosporium spp.were always isolated from each bach of persimmons with high detection rate,while Cladosporium spp.and Alternaria spp.were always isolated from black rot tissues at top of the fruits.Alternaria spp.has the highest detection rate.3.Pathogenicity tests of pathogens isolated from rot tissues resulted Alternaria tenuissima confirmed by morphological characterization,rDNA-ITS analysis and endopolygalacturonase gene analysis was main cause of black rot at the top of the fruit.The main pathogen in black rot granules was confirmed to be Cladosporium spp.by morphological characterization and rDNA-ITS analysis.