Biological control of Sclerotium cepivorum Berk. (Onion White Root Rot) using Trichoderma koningii Oudem
Creators: name: Metcalf, Dean Andrew
Publisher: name: University Of Tasmania
Year: 2023

description: At the time this study commenced Trichoderma koningii strains had reduced incidence of Sclerotium. cepivorum infection of onions in field trials by 60%. The goal of the work was to try to improve T. koningii 's efficacy. To gain some understanding of the antagonism process histological and enzymatic studies of antagonism within infected onion roots were undertaken as well as ecological studies and field trial evaluation. 'Histological studies confirmed.S cepivorum hyphae penetrated the epidermis of onion roots and grew into the hypodermis and cortex. In early stages of the infection only cells S. cepivorum grew through were lysed, as the infection developed cells were killed, and cell walls disintegrated in the zone ahead of the infection hyphae. The root epidermis and stele tissues were more resistant to hydrolysis than the cortex, resulting in formation of a cavity filled with S. cepivorum hyphae within the cortex. S. cepivorum was shown to produce three isozymes of polygalacturonase and three isozymes of pectinesterase in infected tissue. A series was noted in enzyme production with pectinesterases produced first on onion cell wall substrate, followed by polygalacturonase. A novel technique for localisation of pectolytic enzymes in infected tissue was developed by loading tissue segments into wells of an electrophoresis gel. Using this technique it was confirmed that distribution of pectolytic enzymes which diffused ahead of infection hyphae were correlated to cell wall dissolution. It was postulated that S. cepivorum may derive advantage from the resistance of the epidermis to hydrolysis, which may serve as a barrier to secondary invaders which may compete for nutrients or inhibit S. cepivorum. When placed on the epidermis of healthy onion roots T. koningii (Tr5) was observed to grow in the epidermal mucilage without entering healthy epidermal tissue. When T. koningii was placed on the epidermis of S. cepivorum infected roots it was observed to actively colonise epidermal passage cells with little colonisation of other epidermal tissues; before branching and spreading throughout the infected or damaged tissues below. Passage cells appear to exhibit some differences in suberisation, and possibly lignification to other epidermal cells. Electrophoretic studies showed that T. koningii produced one polygalacturonase and two pectinesterase in liquid culture, and produced pectinases in damaged onion root tissues. Changes were observed in S. cepivorum hyphae when T. koningii colonised infected tissue, including detachment at the septa, dissolution of cell walls, and lysis of hyphal apices with release of the protoplasm. Contact between hyphae was not necessary for this lysis to occur. An electrophoresis protocol developed in this study showed that T. koningii produced chitinolytic enzymes likely to be component of the antagonism process. The T. koningii chitinase complex consisted of at least four proteins, two endochitinases and two chitobiases. Electrophoresis of root segments in which lysis of S. cepivorum hyphae had occurred showed that T. koningii produced at least two chitinolytic enzymes (an endochitinases and a chitobiase) in these tissues. T. koningii was able to produce chitinolytic enzymes to use S. cepivorum sclerotia as a sole source of nutrition. Enzymes produced in degradation of crustacean chitin were the same ones produced to degrade S. cepivorum cell walls, which has useful implications for soil amendments. Pot trials where T. koningii was added as a continuous band of inoculum just below germinating seeds, demonstrated that when T. koningii was well established in the rhizosphere it was able to prevent at least 82% of infections initiated 3cm below the onion base plate. A number of field amendment methods were investigated including fluid drill sowing of seed with living T. koningii mycelium and in furrow spore sprays, along with s.olid carriers including crabshell chitin, sawdust, and a peat/chitin/osmocote blend. To monitor establishment in the rhizosphere a selective medium (RASP) was developed, which was used in combination with isozyme profiles to distinguish Trichoderma isolates growing on onion roots. Studies of rhizosphere establishment suggested that Tr5 had a poor ability to become established in soils of pH 7.5, however at pH 5.5 a high proportion of roots were colonised by Tr5. S. cepivorum infection is .generally most severe in Tasmania when soil temperatures are in the 11 to 15¬¨‚àûC range. Studies of the effects of soil temperature on biocontrol demonstrated that Tr5 was more able to suppress infections when soil temperature was 10 to 12¬¨‚àûC than 15 to l8¬¨‚àûC. When Tr5 was well established in the rhizosphere a consistent ability to suppress between 63 to 79% of infections in soils with S. cepivorum sclerotial densities ranging from 10 to 100 sclerotia per kilogram was demonstrated. The ability of S. cepivorum sclerotia to infect the onion base plate decreased with increasing depth of burial, and Tr5 was further able to suppress a greater proportion of infections originating from a depth of 7cm than 4cm. ¬¨‚àëThis finding may have implications for integrated control, as sclerotia near the soil surface may be more readily stimulated to germinate by sclerotial germination stimulants, and integration of the two measures will be a subject of future work.