Preliminary Study On The Inhibitory Effect Of Soil Antimicrobial Compounds On Proteins Of Spore Germination

As plant-parasitic nematodes cause a surprising amount of losses to agriculture, biological control of plant-parasitic nematodes has been in a hot research. However, in a practical application, soil fungistasis seriously affects the germination and growth of biocontrol fungus spores in the soil, which is one of relatively low and unstable critical factors of biocontrol agents control efficiency. Previous studies have shown that as the major inhibitory factor in the soil, the volatiles, such as benzaldehyde and ammonia, can suppress numerous fungal spores germination and mycelial growth, but the specific molecular mechanisms have not been uncovered so far. Thus, the present study was to investigate the molecular mechanisms of Arthrobotrys oligospora conidial germination suppressed by benzaldehyde and ammonia. Firstly, taking Arthrobotrys oligospora conidia as the research material, the associated protein samples were extracted from ungerminated conidia or germinated for 24h under normal condition, ungermination conidial samples inhibited by benzaldehyde or ammonia. And then the research methods of iTRAQ-HPLC-MS/MS were performed to analyse the differential proteins of the samples. Finally, based on the analysis on the differential proteins, including protein species, functions, signaling pathways and metabolism pathway, this study attempts to elucidate the molecular mechanisms of Arthrobotrys oligospora conidial germination inhibited by the soil inhibitory compounds and Arthrobotrys oligospora responding to soil fungistasis from the proteomics perspective.The main results of this paper are:1. Base on the iTRAQ-LC-MS/MS analysis and the MaxQuent search results, a total of 3087 proteins were identified in the benzaldehyde treatment group, among of which 2669 proteins were quantified. And 3100 proteins were totally identified in the ammonia treatment group, among of which 2559 proteins were to perform quantitative analysis.2. To select differentially expressed proteins, the cutoff value for up-regulated proteins was 2.0-fold and for down-regulated proteins was 0.5-fold. The results were that 1) in the benzaldehyde sectionalization, ungerminated conidia being served as control,147 proteins were up-regulated and 200 proteins were down-regulated in the normal 24h-germinating treatment; 171 proteins were up-regulated and 118 proteins were down-regulated in the benzaldehyde treatment group.2) in the ammonia sectionalization, ungerminated conidia being served as control,155 proteins were up-regulated and 179 proteins were down-regulated in the normal 24h-germinating treatment; 101 proteins were up-regulated and 316 proteins were down-regulated in the ammonia treatment group.3. Through the comparison of the differentially expressed proteins in normal 24h-germinating treatment group and benzaldehyde treatment group, we found that 52 up-regulated proteins and 28 down-regulated proteins were shared in both groups; 112 up-regulated proteins and 82 down-regulated proteins were alone influenced by benzaldehyde. Similarly,74 up-regulated proteins and 281 down-regulated proteins were alone influenced by ammonia.4. Based on the functional analysis of up-/down-regulated proteins of samples suppressed by benzaldehyde or ammonia, the results were that the 112 unique up-regulated proteins of the sample suppressed by benzaldehyde were grouped into as many as 22 subcategories, involving mainly in the clusters’general function prediction only’,’amino acid transport and metabolism’n’posttranslational modification, protein turnover, chaperones’, representing 19.84%,10.32%,8.73%, respectively; the 82 unique down-regulated proteins of the sample were classified into 19 subcategories such as’translation, ribosomal structure and biogenesis (20.21%)’,’intracellular trafficking, secretion, and vesicular transport (10.46%)’,’posttranslational modification, protein turnover, chaperones (9.57)’and so on.5. Similarly, according to the functional analysis of up-/down-regulated proteins of sample suppressed by ammonia, we found that the 74 unique up-regulated proteins of the sample inhibited by ammonia were grouped into as many as 16 subcategories, involving mainly in the clusters’general function prediction only’、’Energy production and conversion’、’posttranslational modification, protein turnover, chaperones’, representing 22.81%、14.04%、12.28%, respectively; the 281 unique down-regulated proteins of the sample were classified into 24 subcategories such as’general function prediction only (11.72)’、’translation, ribosomal structure and biogenesis (9.59%)’、’posttranslational modification, protein turnover, chaperones (8.59%)’and so on.6. The GO analysis of unique up-/down-regulated proteins of samples suppressed by benzaldehyde or ammonia were performed to do.7. On the basis of bioinformatics analysis and related documents search, we held the view that benzaldehyde caused the down-regulated expression of some important proteins, such as the translation initiation factors, elongation factors, seven subunit ribosomal proteins, Ran protein and a conserved protein associated with cAMP, which might be the main reason of benzaldehyde suppression on Arthrobotrys oligospora ATCC24927 conidial germination. As for the sample suppressed by ammonia, we speculated that ammonia caused the down-regulated expression of some important proteins, such as mitotic kinesin, three cell division control proteins, seven aminoacyl-tRNA synthetase like valine tRNA synthetase, elongation factors, seven subunit ribosomal proteins, five mitogen-activated protein kinases(MAPK), ras protein and G protein a subunit, which might be the main reason of ammonia suppression on Arthrobotrys oligospora ATCC24927 conidial germination.8. Based on the bioinformatic analysis and current literatures, the conidia might primarily enhance the stress resistance to benzaldehyde by up-regulating the gese expression of phosphate carboxylase kinase, lactoylglutathione lyase and subtili sin-like protease. While conidia mainly up-regulated superoxide dismutase, thioredoxin peroxidase, lactoylglutathione lyase to response to chemical stimuli, and enhanced resistance by strengthening the reactive oxygen scavenging mechanism; besides, malate dehydrogenase provided energy for conidia to ensure physiological activities associated with resistance, thereby participated in the defense mechanisms of resistance to chemical stimulation.