The Pathogenic Molecular Differences Of Setosphaeria Turcica F. Sp. Zeae And Setosphaeria Turcica F. Sp. Sorghi

Setosphearia turcica which causing leaf blight of corn, sorghum and other gramineous crops, is an important phytopathogenic fungi. Setosphearia turcica was divided into Setosphearia turcica f. sp. zeae and Setosphearia turcica f. sp. sorghi. Incompatible host of two formae speciales can not be infected by incompatible pathogens. The pathogenic specialization demonstrated pathogenic differences between the formae speciales of S. turcica. At present, no researchs on the pathogenic molecular mechanism and genetic basis of the formae speciales of S. turcica were reported. The study researched on the pathogenic molecular mechanism in the interaction progress of host-formae speciales and relative differential pathogenic genes causing pathogenic specialization. It has an important theoretical significance for revealing the pathogenic specialization of formae speciales of S. turcica. Main results in this studyr were as follows:1. S. turcica f. sp. zeae and S. turcica f. sp. sorghi were widely dispersed among the regions where maize and sorghum are cultivated in China. The strict pathogenic differences and genetic diversity existed between two formae speciales of S. turcica.Experiments were conducted to using morphological identification, ITS and pathogenicity test and UP-PCR analysis. Main results in this study were as follows. (1)The geographical distributions of S. turcica f. sp. zeae were more widely than S. turcica f. sp. sorghi because of cultivated areas’limitations of sorghum in China. Therefore, pathogenicity was no directly correlated with the geographic distributions in the formae speciales. (2) After inoculation of S. turcica f, sp. zeae, the maize’s blades were covered with fusiform necrotic spots, and the incompatible reactions observed on sorghum and Sudan grass were fuchsia-colored flecks. After inoculation of S. turcica f. sp. sorghi, the long fusiform necrotic spots with light brown centers and fuchsia-colored margins were formed on the blades of the sorghum and Sudan grass, and incompatible reactions appeared symptomless on maize. (3) UP-PCR was useful for the relationship of pathogenic specialization and genetic relations among the formae speciales of S. turcica. But it did not indicate a correlation between and genetic diversity and geographic distribution. The isolates of each clade belonged to the same forma specialis, and different formae speciales were assigned to different clades. It was consistent with the results of pathogenicity tests and geographical distribution of two formae speciales. Therefore, UP-PCR was useful for the study of genetic relations among the formae speciales of S. turcica and reveals a high level of genetic diversity among these pathogens.2. The differences and similarities of biological phenotype existed between two formae speciales of S. turcica.Experiments were conducted to using biological characteristics observation, physiological and biochemical indicators detection and RT-PCR. (1) The differences and similarities of biological phenotype were revealed under the conditions which included different culture medium, nitrogen source, carbon source, illumination, temperature, pH and humidity. (2) The differences and similarities of biological phenotype were also showed in the aspect of physiological and biochemical indicators and genes, such as appressorium formation, HT-toxin and composition of secondary metabolites, melanin synthesis, cell wall synthetase, cell wall degradation enzymes, laccase and hydrophobic.3. The interacted infection mechanism differences existed between two formae speciales of S. turcica.Experiments were conducted to using infection phenotypic comparison, optical microscopy observation, SEM, TEM, HT-toxin biological activity detection, physiological and biochemical indicators detection. The results were showed as follows. (1) The forma specialis can normally infect its compatible host with typical symptoms. (2) The forma specialis only invade its incompatible host but no typical symptoms. In the cell observation, the pathogens were limited to the chloroplasts which the normal functions of chloroplasts were maintained. In the aspect of cell structure change, the resistant structure were presenced in the plant cells, such as folding cell wall, thickening cell wall, closely arranged electron dense material and amorphous structures to resist pathogens. (3) When the formae speciles infected compatible or incompatible hosts, the pathogenic mechanical of HT-toxins was on the basis of mechanical damage. No significant effect was showed on the incompatible host. (4) It showed that the increasing activity of CWDE and the decreasing chlorophyll content in the compatible interaction. It showed the fluctuations and the stationary of the activity of C WDE and chlorophyll content with disease extension.4. The differences of the transcriptomes existed in the interaction of host-forma specialis.Experiments were conducted to using RNA-seq, qRT-PCR, anslysis of GO and KEGG Pathway. The results were shown as follows. (1) The qRT-PCR results were consistent with RNA-seq. It proved the accuracy of the transcriptomes’results. (2) The differences of genes’numbers and types were revealed in the interaction transcriptomes of two formae speciales. The numbers which were in differential genes, up-regulated genes, down-regulated genes and specific genes in StSCK vs StSIN were less than in StZCK vs StZIN. For 48h after inoculation, in the StSCK vs StSIN and StZCK vs StZIN, respectively, there were 1132 and 2549 of the differential expression genes,399 and 1816 of the special expression genes and 773 of common differential expression genes. With the fold changes over 5,47 up-regulated genes were in the StSCK vs StSIN. With the fold changes over 2.5,61 up-regulated genes were in the StZCK vs StZIN. The related pathogenic biological processes and functions of the differential expression genes were similar, including hydrolase activity, oxidoreductase activity, carbohydrate metabolism and histidine protein kinase activity and so on. (3) The genetic types and functions of differential expression genes were different in GO enrichment. Carbohydrate metabolic process, plant-type cell wall organization or biogenesis and hydrolase activity were included in StSCK vs StSIN, which belonged to biological processes, molecular function and cellular components. It was divided into biological processes and cellular components in StZCK vs StZIN, without significant GO enrichment. (4) The genetic numbers and types of differential expression genes were different in KEGG pathways. In the StSCK vs StSIN and StZCK vs StZIN, respectively, there were 22 and 35 of the KEGG Pathways. In the StSCK vs StSIN, the related pathogenic pathways included peroxidase, starch and sugar metabolism, secondary metabolites biosynthesis, cytochrome P450 and protease. In the StZCK vs StZIN, the related pathogenic pathways were presented, such as, starch and sucrose metabolism, fatty acid degradation, microbial metabolism in diverse environments, proteasome, glutathione metabolism and MAPK signaling pathway.5. The differential expressions of related pathogenic genes existed in the different times.Experiments were conducted to using qRT-PCR. The results were showed as following. (1) In the results of hydrolytic enzyme genes, StsPEL8, StsPEL9, StsBGL5 and StzPEL5 all revealed rich expression level. Since 48 h, the genes’ expression rise sharply and the times of high expression were different. It showed that hydrolytic enzyme genes contributed to degrading cell wall, promoting the extension of hyphae and development of disease. (2) In the results of oxidordeuctase genes, the genes all revealed rich expression level. The genes’ expression showed the times of high expression were different. It showed that hydrolytic enzyme genes contributed to degrading cell wall, promoting the extension of hyphae and development of disease. (3) In the results of oxidordeuctase genes, the genes all revealed rich expression level. The genes’ expression showed the times of high expression were different. StsPDE12、StsPKC13 and StsPKC17 were up-regulated expression during course of disease. StzSte20 was up-regulated expression in 6 h and down-regulated expression in other times. StsHMGCL and StzHogl were down-regulated expression in 48 h and up-regulated expression in other times. StzGlol、StzHsp72 and StzPKC15 were up-regulated expression.6. The functions of pathogenic difference gene were predicted.Experiments were conducted to using cloning and function prediction. The results were showed as follows. It was consistent with RNA-seq results and cloning results. (1) In the results of DNA cloning, StsACOXl and StzCypl also existed in two geneome of S. turcica f. sp. zeae and S. turcica f. sp. sorghi. In the cDNA of 48h’s interactive samples cloning, StsACOXl and StzCypl existed respectively in S. turcica f. sp. zeae and S. turcica f. sp. sorghi. It revealed that the specific expressions of StsACOXl and StzCypl only presented in the compatible interaction. (2) 368 amino acids were encoded by StsACOXl. The molecular weight was 43.3 kD. The theoretical isoelectric point (pl) was 9.88. The molecular formula was Ci892H283oN5980527S25. The StsACOXl protein did not located in the biofilm and speculated intracellular proteins. The protein belonged to the ANK gene families. The high homology was showed between StsACOX1 and the protein of S. turcica Et28A. StsACOXl were up-regulated expressions, respectively in 72 h. (3) 501 amino acids were encoded by StsCypl. The molecular weight was 55.8 kD. The theoretical isoelectric point (pl) was 9.24. The molecular formula was C2451H3916N694O725S34. The StzCypl protein did not located in the biofilm and speculated intracellular proteins. The protein belonged to the Cyp 450 gene families. The high homology was showed among StzCypl and the protein of S. turcica Et28A, B. maydis and B. zercola. StzCypl were up-regulated expressions, respectively in 120 h.