Disease Differences Of Colletotrichum Truncatum On Resistant And Susceptible Soybean Lines And Characterization Of A Mapk Gene CTPMK1in The Pathogen

Glycine max is an important cash crop and occupies a significant position in agricultural production system of China. Soybean anthracnose caused by Colletotrichum truncatum (Schwein.) Andrus&W.D.Moore is one of the important diesases in soybean production areas of the world. It can cause severe yield losses. Consequently, the prevention and curing of anthracnose is always an serious problem in soybean production. In this study, soybean anthracnose pathogens were isolated and identified from soybean-producing areas, then the identification of resistant soybean lines, the interaction between soybean and C. truncatum, and the fungi development and its pathogenicity related molecular mechanisms were studied. It builds basis for both practise and theory to understand and master the discipline of occurrence and development of this disease, with which we can take effective measures to control.1. Samples of suscepted soybean anthracnose were collected from soybean-producing areas in Taixing and Jiangpu farm of Jiangsu province.41pathogens were obtained after isolation, purification and pathogenicity experiments. The morphology of these pathogens could be divided into two categories. One was similar to C. truncatum, and the colony was regular round-like and had neat edges. It also had loose mycelia, of which young was white and aging was gray. The other colony had not been reported. It was irregular round, jagged and had less aerial mycelia, of which the central was white and the fluffy was showing bright red-brown. One pathogenic strain from each category was selected to sequence the ITS (internal transcribed spacer). Both the sequences reached100%with C. truncatum. Based on the morphological characteristics and the sequences of ITS, the two categories strains were identified as C. truncatum for the following research.2. The choosing and using of resistant cultivars is an effective and economical measurement to combat with soybean anthracnose. Making clear the resistance of soybean germplasm to soybean anthracnose is a precondition of resistance breeding. In this study, we identified the resistance to anthracnose of59Zhejiang soybean lines, using the method of dripping inoculated leaves with spore suspensions. The two representative anthracnose strains named JS11-32and TB8-15identified before were chosen for the test. We found that96.61%of soybean lines showed resistance to strain JS11-32while55.93%to TB8-15. Besides, all the resistance lines to TB8-15were resistant to JS11-32. We could make a conclution that Zhejiang soybean lines showed a good performance to anthracnose. It is deserved to make use of some resistant species in field practise.3. We compared the infection differences of anthracnose pathogen on resistant soybean species5D068(showing incompatible interaction) and susceptible one Hefeng47(showing compatible interaction) by Trypan Blue dying experiment. We found that for both cultivars, conidial germination and appressoria formation were observed within24hpi. The infection hyphae were appeared in susceptible cultivar leaves at48hpi, while the infected epidermal cells of resistant one were necrotic showing characteristic features of hypersensitive cell death. We discovered that resistant cultivar produced more prominent reactive oxygen species under appressoria in infected plant cells and even the border of surrounding uninfected plant cells during the early infection period by diaminobenzidine (DAB) staining. In contrast, in the susceptible ones, the DAB-positive reaction was just a little evident around appressoria.4. MAPK signaling pathway is significantly conserved in eucaryotic organisms. It plays a very important role in perception of stimulus, growth and development, conidia production and germination of fungi. According to the conserved MAPK sequences of C. gloeosporioides and C. higginsianum, of which the whole genome sequences have been published, a gene designated CtPMKl was identified in C. truncatum. It encodes a protein of355amino acids and has the typical characteristics of MAPK, which has11protein kinase domains and a kinase activation site of sequence TEY. The deletion of CtPMKl from C. truncatum resulted in reduced mycelia growth, decreased melanin production and sporulation. Pathogenicity assay indicated that the CtPMK1deletion mutant lost the ability to invade in soybean leaves. Onion epidermis penetration observation showed that the conidia of mutants could not form appressorium and the hyphal tips of mutant faied to form appressorium-like structures. In conclusion, the above results indicated that CtPMKl was responsible for the pathogenicity via regulating the appressorium and appressorium-like structures production in C. truncatum. The Magnaporthe oryzae PMK1gene could complement phenotypes and pathogenicity of CtPMK1deletion mutant, which indicated that FUS3-KSS1like gene was conserved in fungi.