Mechanisms Of The Interactions Between Endophytic Fungi Chaetomium Spirale, Stagonospora Sp. And Pathogens Or Host Plant

In recent years, because of singleness cultivar of crops and enhancement of multiple-crop index, considerable losses in agricultural production are due to plant diseases caused by pathogens every year. In general, chemical pesticides can effectively protect plants from many pathogens. But the excessive application of chemical pesticides has not only enhanced the pathogens resistance to fungicides, but also caused environmental contamination and affected the health of human beings. Public concerns about harmful effects of chemical pesticides on the environment and human health, as well as the pathogens have developed resistance to the fungicides have promoted a research for safer control approach. Biocontrol is an environmentally friendly and efficient supplement or alternative to chemical pesticides management. Therefore biocontrol of pathogens has received particular attention as a promising control approach. More and more investigations revealed that many endophytic fungi have very great potential of biocontrol on pant diseases. The key of control plant diseases using endophytic fungi as biocontrol agent is first to understand mechanisms of the interactions between antagonist and pathologens or host plant. In this research, ultrastructure and cytochemistry of hyphal interaction between endophytic Chaetomium spirale ND35 and soil-born plant pathogen Rhizoctonia solani, cell wall degrading enzymes involved in mycoparasitism, interaction between beneficial endophytic Stagonospora sp. 4/99-1 and reed root infected were investigated by light microscopy, scanning and transmission electron microscopy. In addition, role of antibiotics in biocontrol of apple canker was studied by separation and purification of antibiotics and bioassay in the laboratory and application of fermentation of C. spirale ND35 in the field. Antifungal, synergistic interactions between antibiotics and hydrolytic enzymes from endophytic C. spirale ND35 were determined using a pure antibiotic combined with a purified endo-?-1, 3-glucanase in this paper. The main results are listed as follows: 1. The hyphal interaction between endophytic biocontrol agent Chaetomium spirale ND35 and the soilborn plant pathogen Rhizoctonia solani was studied by light microscopy and transmission electron microscopy (TEM), as well as further investigated by gold cytochemistry to assess the potential role of cell wall degrading enzymes (CWDEs) during the mycoparasitic process. Results indicated that the coiling of C. spirale around R. solani and intracellular growth of the antagonist in its host occurred frequently. Moreover, The growth and development of C. spirale were associated with highly morphological changes of the host fungal cell, characterized by retraction of plasma membrane and cytoplasm disorganization. Further TEM investigations through localization by gold immunocytochemistry showed that contact between the two fungi was mediated by an amorphous ?-1,3-glucan-enriched matrix originating from cell wall of the antagonist C. spirale ND35 and sticking to its host surface at sites of potential antagonist entry. However, the antagonist was capable of penetrating this barrier, indicating that ?-1,3-glucanases were produced during the mycoparasitic process. Localization of N-acetylglucosamine residues (chitin) with the gold-labeled WGA implicated that chitinases might be involved in the cell wall degrading of R. solani in this antagonistic process as well. 2. Secretion of hydrolytic enzymes, mainly including glucanases and chitinases, is considered the most important step in the mycoparasitic process. In this study, an about 110 kDa exo-?-1,3-glucanase from C. spirale ND35 was detected both in culture filtrate and directly on PAGE and IEF gels, as well as chitinases, although protease was not detectable on litmus milk agar plates. Coiling and penetrating the hyphae of host fungus Valsa mali were observed by scanning electron microscope (SEM), which may be related to the synergistic interactions between ?-1,3-glucanases and chitinases. The ?-1,3-glucanase activity of C. spirale ND35 varied considerably when C. spirale ND35 was grown in different carbon sources during various incubation time , and might be subjected to both induction by substrate and catabolite repression. 3. Role of antibiotics in biocontrol of apple canker was preliminarily investigated using endophytic fungus Chaetomium spirale strain ND35 in the laboratory, greenhouse and in the field. Results of dual culture on PDA plate demonstrated that C. spirale ND35 showed strongly antagonistic activity against V. mali. The inhibitory zone in the interaction area of colonies both C. spirale ND35 and V. mali was observed after three days inoculation. The culture filtrates, containing antibiotics, of antagonist grown in corn steep powder broth were able to inhibit the mycelial growth of V. mali much more effectively than that of antagonist in malt extract broth (MEB). After C. spirale ND35 in corn steep powder broth was incubated for 12 days, the liquid cultures were filtrated and bioassay was conducted. Inhibitory effect of the crude extract of cultures was showed by the emergence of inhibitory zone and the reduction of pathogenic spore germination rate. Antibiotic production by C. spirale ND35 was separated and purified by extraction of ethyl acetate, dehydration of water-free sodium sulphate, concentration in a rotary evaporator, silica gel filtration chromatography and thin-layer chromatography. A yellow solid product was obtained and its effect of antifungal activity against the pathogen of apple canker was also tested in vitro. Results of biocontrol in greenhouse and in the field indicated that the incidence of apple canker of the treatment with C. spirale ND35 was significantly lower than other mode of treatments, including treatment with V. mali alone, and water as control. The reisolation experiment suggested that C. spirale ND35 was successfully able to colonize in stems and branches of apple trees. And the Chaetomium colonization rate of the treatment with solid wheat bran culture was at higher level when compared with that of the treatment with liquid culture of antagonist grown incorn steep powder broth. But the biocontrol effect of liquid culture was better than that of the solid culture in the field. Results demonstrated that antibiotics production by C. spirale ND35 play an important role in controlling apple canker. The present research supplied the evidence for the potential of C. spirale strain ND35 in biocontrol of Valsa canker of apple tree. 4. Antifungal, synergistic interactions between antibiotics and hydrolytic enzymes from endophytic Chaetomium spirale ND35 demonstrated that the crude extract of culture filtrate from the C. spirale ND35 grown in the SM medium containing cell wall preparation (CWP) of Rhizoctonia solani was strongly inhibitory to mycelial growth and conidial germination of different phytopathogenic fungi including Valsa sordida, V. mali, Glomerella cingulata and Curvulavia lunata. The crude extract of culture filtrate contained the activity of ?-1,3-glucanase (endo-glucanase and exo-glucanase) and chitinase. Enzymic activity reached 0.19 U and 0.09 U, respectively. The crude extract of culture filtrate, containing antibiotics, from C. spirale ND35 grown in 3% corn steep powder broth also inhibited sporangial germination and mycelial growth of these pathogens. The inhibitory effect of combination both a purified endo-?-1,3-glucanase (GLUC73) and a pure antibiotic from C. spirale ND35 on the conidial germination and germ tube elongation of G. cingulata was tested in vitro. When antibiotic and endo-?-1,3-glucanase were applied together, a synergistic inhibitory effect was observed. Applied alone, 1.0 μg ml-1 of antibiotic or 40 μg ml-1 of endo-?-1, 3-glucanase had no or only a very low inhibitory effect. Their combination resulted in about 78% inhibition of spore germination. Even as low an amount of endo-?-1,3-glucanase as 10 μg ml-1 increased the effect of 1.5 μg of antibiotic per milliliter from lower than 25 to over 50% inhibition. In addition, 80 μg of endo-?-1,3-glucanase per milliliter dramatically increased the inhibitory activity of 1.0 μg of antibiotic per milliliter from 0 to 90%. Synergistic antifungal activity of antibiotics and hydrolytic enzymes (endo-?-1,3-glucanase) may play an important role in biological control by endophytic C. spirale ND35. 5. Stagonospora sp. (4/99-1) is a beneficial endophytic fungus transmitted by seeds of Phragmites australis. This fungus also can penetrate the root epidermis as an alternative route to become established in the host. Hyphae attracted by the root proliferated on the root surface, preferably over the anticlinal walls. Penetration occurred directly by undifferentiated hyphae or was supported by hyphopodia. Within the root, hyphal growth was restricted to the walls of the outermost two cortical cell layers. Further ingress into the lumen of epidermal or cortical cells was blocked by papillae. In the rare cases the fungus managed to penetrate into cortical cells, the plant cell reacted with a hypersensitive response. Antibodies raised against Stagonospora sp. (4/99-1) revealed fungal material in the host apoplast and close to the plasmalemma. Attachment to the plant membrane or uptake of fungal exudates by the host cell may explain plant defence reactions. Root penetration of Stagonospora and restricted development in the plant obviously served as an alternative to keep young seedlings infected.