Biological control of Sclerotium cepivorum Berk. (Onion White Root Rot) using Trichoderma koningii Oudem

摘要

At the time this study commenced Trichoderma koningii strains had reducedudincidence of Sclerotium. cepivorum infection of onions in field trials by 60%. Theudgoal of the work was to try to improve T. koningii 's efficacy. To gain someudunderstanding of the antagonism process histological and enzymatic studies ofudantagonism within infected onion roots were undertaken as well as ecological studiesudand field trial evaluation.ud'Histological studies confirmed.S cepivorum hyphae penetrated the epidermis ofudonion roots and grew into the hypodermis and cortex. In early stages of the infectionudonly cells S. cepivorum grew through were lysed, as the infection developed cellsudwere killed, and cell walls disintegrated in the zone ahead of the infection hyphae. Theudroot epidermis and stele tissues were more resistant to hydrolysis than the cortex,udresulting in formation of a cavity filled with S. cepivorum hyphae within the cortex.udS. cepivorum was shown to produce three isozymes of polygalacturonase and threeudisozymes of pectinesterase in infected tissue. A series was noted in enzymeudproduction with pectinesterases produced first on onion cell wall substrate, followedudby polygalacturonase. A novel technique for localisation of pectolytic enzymes inudinfected tissue was developed by loading tissue segments into wells of anudelectrophoresis gel. Using this technique it was confirmed that distribution ofudpectolytic enzymes which diffused ahead of infection hyphae were correlated to celludwall dissolution. It was postulated that S. cepivorum may derive advantage from theudresistance of the epidermis to hydrolysis, which may serve as a barrier to secondaryudinvaders which may compete for nutrients or inhibit S. cepivorum. When placed on the epidermis of healthy onion roots T. koningii (Tr5) wasudobserved to grow in the epidermal mucilage without entering healthy epidermal tissue.udWhen T. koningii was placed on the epidermis of S. cepivorum infected roots it wasudobserved to actively colonise epidermal passage cells with little colonisation of otherudepidermal tissues; before branching and spreading throughout the infected or damagedudtissues below. Passage cells appear to exhibit some differences in suberisation, andudpossibly lignification to other epidermal cells. Electrophoretic studies showed that T.udkoningii produced one polygalacturonase and two pectinesterase in liquid culture, andudproduced pectinases in damaged onion root tissues. Changes were observed in S.udcepivorum hyphae when T. koningii colonised infected tissue, including detachmentudat the septa, dissolution of cell walls, and lysis of hyphal apices with release of theudprotoplasm. Contact between hyphae was not necessary for this lysis to occur. An electrophoresis protocol developed in this study showed that T. koningiiudproduced chitinolytic enzymes likely to be component of the antagonism process. TheudT. koningii chitinase complex consisted of at least four proteins, two endochitinasesudand two chitobiases. Electrophoresis of root segments in which lysis of S. cepivorumudhyphae had occurred showed that T. koningii produced at least two chitinolyticudenzymes (an endochitinases and a chitobiase) in these tissues. T. koningii was able toudproduce chitinolytic enzymes to use S. cepivorum sclerotia as a sole source ofudnutrition. Enzymes produced in degradation of crustacean chitin were the same onesudproduced to degrade S. cepivorum cell walls, which has useful implications for soiludamendments.udPot trials where T. koningii was added as a continuous band of inoculum justudbelow germinating seeds, demonstrated that when T. koningii was well established inudthe rhizosphere it was able to prevent at least 82% of infections initiated 3cm below theudonion base plate. A number of field amendment methods were investigated includingudfluid drill sowing of seed with living T. koningii mycelium and in furrow sporeudsprays, along with s.olid carriers including crabshell chitin, sawdust, and audpeat/chitin/osmocote blend.udTo monitor establishment in the rhizosphere a selective medium (RASP) wasuddeveloped, which was used in combination with isozyme profiles to distinguishudTrichoderma isolates growing on onion roots. Studies of rhizosphere establishmentudsuggested that Tr5 had a poor ability to become established in soils of pH 7.5,udhowever at pH 5.5 a high proportion of roots were colonised by Tr5. S. cepivorumudinfection is .generally most severe in Tasmania when soil temperatures are in the 11 toud15°C range. Studies of the effects of soil temperature on biocontrol demonstrated thatudTr5 was more able to suppress infections when soil temperature was 10 to 12°C thanud15 to l8°C.udWhen Tr5 was well established in the rhizosphere a consistent ability toudsuppress between 63 to 79% of infections in soils with S. cepivorum sclerotialuddensities ranging from 10 to 100 sclerotia per kilogram was demonstrated. The abilityudof S. cepivorum sclerotia to infect the onion base plate decreased with increasinguddepth of burial, and Tr5 was further able to suppress a greater proportion of infectionsudoriginating from a depth of 7cm than 4cm. ·This finding may have implications forudintegrated control, as sclerotia near the soil surface may be more readily stimulated toudgerminate by sclerotial germination stimulants, and integration of the two measuresudwill be a subject of future work.