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

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Metcalf, Dean Andrew (1997) Biological control of Sclerotium cepivorum Berk. (Onion White Root Rot) using Trichoderma koningii Oudem. PhD thesis, University of Tasmania.

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Abstract

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°C 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°C than
15 to l8°C.
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. ·This 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.

Item Type: Thesis (PhD)
Keywords: Trichoderma, Sclerotium cepivorum, Onions
Copyright Holders: The Author
Copyright Information:

Copyright 1997 the Author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s)

Additional Information:

Thesis (Ph.D.)--University of Tasmania, 1998. Includes bibliographical references

Date Deposited: 03 Feb 2015 03:03
Last Modified: 11 Mar 2016 05:55
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