Infection Characteristics Of Two Colletotrichum Fructicola Strains With Distinct Symptoms On Pear Leaves

In recent years,the early abnormal defoliation occurred on sandy pear(Pyrus pyrifolia(Burm.f.)Nakai)in southern China,and caused a great economic loss.In our laboratory,it was systematically identified and confirmed that Colletotrichum fructicola was causing this disease.At the same time,there are two different types of C.fructicola in the field.Type I produce non-expanding black spot symptoms,and type II produce expandable necrotic symptoms.In this study,we selected representative strain FJ-85(producing black spot symptoms)and strain FJ-11-2(producing necrotic lesions)derived from the two different symptom types.We compared the two types of representative strains,by compared their sexual and asexual performance on the impact of symptoms,and were used fluorescence labeling and tissue staining to observe the infection process and to provide a new experimental basis for exploring the pathogenic mechanism and the pathogenic diversity of the C.fructicola in pears in China.The main results were as follows:1.Studies on the biological characteristics and pathogenicity for the monospora strains from ascospores of C.fructicola in pear of two different pathogenic strains.Two pathogenic strains including FJ-85(producing black spot)and FJ-11-2(producing necrotic lesion)of Colletotrichum fructicola in pear were studied.The results showed that these two strains could produce plus and minus monospora strains by single ascospore separating and pure culturing.The plus monospora strains formed light white colonies and their single ascospores produced plus and minus monospora strains.The minus monospora strains formed dark gray colonies and their single ascospores only produced minus monospora strains.Of the monospora strains from these two strains,the proportions of the minus types were significantly higher than the plus ones.By paired culture with two types of monospora strains,the perithecia were observed in their colonies as well as at the junction of two colonies.However the perithecia were observed only in their colonies throught paired culture with the same type of monospora strain.The results showed that there was homothallism in the same monospora strains,while there was still heterothallism between plus and minus monospora strains.The pathogenicity of the plus monospora strains were similar to that of the original strains,while the minus monospora strains were weaker than the original ones.The symptoms caused by two types of monospora strains on the leaves of Pyrus pyrifolia var.Cuiguan were the same as those of their original strains.2.Establishment of GFP transformation system of two different pathogenic strains.The GFP labeling of two different pathogenic strains by utilizing ATMT method.Selection and determination of fluorescence stability and hygromycin B resistance of transformants.And detecting the hph and gfp genes of transformants by molecular methods.a And then comparison the wild strain with transformants by colony morphology,growth rate,and virulence,respectively.The transformant FJ-85-24 and FJ-11-2-39 was screened out with not significantly changed.3.Microscopic observation on the infection of pear leaf by GFP transformants of two different pathogenic strains.The microscopical observation was performed regularly after inoculation with conidia suspension of the transformant FJ-85-24 and FJ-11-2-39 on Cuiguan pear leaves with unwound and acupuncture.It was found that strain FJ-85-24 could produce black spots on leaves,but the fluorescence phenomenon was not obvious under fluorescence stereomicroscope.The strain FJ-11-2-39 was no obvious symptoms at early vaccination.Through the fluorescence microscope observation at different times of infection,we found that appressoria attached to leaf surface of black spot after inoculation of strain FJ-85-24,and there was also observed defensive reaction in the surrounding cells of black spot.However,the expansion of mycelium was not observed inside the leaves.We suspected that the symptom of black spot may be caused by the incompatible interaction between some secondary metabolites produced by the appressorium and the host during infection.The strain FJ-11-2-39 was latent infection on the leaves by appressoria.At the late stage of the disease,both strains formed a large number of perithecia under the stratum corneum of leaves.The conidia suspension of the transformant FJ-85-24 and FJ-11-2-39 were inoculated on the healthy "Hosui" pear leaves by non-injury and stab-inoculation.The symptoms appeared significantly earlier,and the expansion of mycelium can be seen in the necrotic areas.The necrosis tissue between palisade tissue and spongy tissue was observed under fluorescence microscope,but no expansion of mycelium at the diseased tissue was observed.4.Observation on the tissue staining of pear leaves infected by the two different pathogenic strains.The result of the observation about the infection process of the two strains showed that the conidial germination rate of strains FJ-85 and FJ-11-2 were similarity,however,there was a significant difference in the formation rate of appressoria at the early stage.The strain FJ-85 mainly formed the appressoria with short germ tube,while the strain FJ-11-2 mainly extended the germ tube.Both strains formed large numbers of appressoria and blastic conidia at the end of the germ tube and surrounding area after 24 hour post inoculation.