Studies on the application of biocontrol agents for the control of seedling diseases.
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Date
2002
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Abstract
The controlled environment of greenhouses, the high value of crops, and the limited number
of registered fungicides offer a unique niche for the biological control of plant diseases.
During the past ten years, over 80 biocontrol products have been marketed worldwide. A
large percentage of these that have been developed in greenhouses could predominate over
chemical pesticides in the same way that biological control of greenhouse insects
predominated in the United Kingdom.
A review of the literature was undertaken to obtain information on biocontrol agents with
specific reference to Trichoderma and Gliocladium spp. Literature on the application, types of
formulations, limitations in formulation, registration and commercialization of these
biocontrol agents were obtained.
Trichoderma harzianum Strain KMD has been used successfully as a biological control agent
against several soil-borne plant pathogens. Biological control agents should possess several
desirable characteristics, including, ease of preparation and application, stability during
transport and storage, abundant production of viable propagules and good shelf-life.
A strain of Trichoderma harzianum KMD with potential biocontrol activity was used to
determine the effect of culture conditions on spore shelf-life. The influence of four growing
media were investigated on the spore ultrastructure and shelf-life, using a basal salts medium
with C:N ratios of 3 and 14, and pH's of 4.0 and 7.0. Mycelial development and sporulation
were positively affected by acidic conditions (pH 4.0). The effect of these culture parameters
on viability and shelf-life were evaluated by counting colony forming units (c.f.u) before and
after seven days of storage at 75% relative humidity (rH): The effect of carbon concentration
on spore viability after seven days of storage was also determined by increasing
concentrations of glucose while a constant C:N ratio of 3 or 14 at pH 4.0 was maintained at a
75% rH. Increasing carbon concentration and C:N ratios increased spore production times.
Spore viability was greatest when harvested from a medium with a C:N of 14 at pH 4.0 even
when storage time was increased to 45 days and rH was reduced to 12%. Ultrastructural
studies showed that spores had two cell wall layers, with the outer being more electron-dense
than the inner layer. This layer is the spore's first defense against adverse conditions. Spores
obtained from this medium were larger, germinated better and had a longer shelf-life than
spores from C:N 3 medium, possibly because the two cell wall layers acted as a thicker
barrier against adverse conditions. Increasing carbon concentration, while maintaining a
constant C:N ratio of 3 or 14 at pH 4.0 slowed down spore production. Viability of spores
were similar when introduced on media with variable carbon concentrations but fixed C:N
ratios. The ultrastructural differences and shelf-life studies, confirmed empirical results from
liquid fermentation studies, that the pH and C:N ratio of the medium upon which spores of T
harzianum KMD strain KMD were produced have critical effects on physical and chemical
structure of the spores and viability. This, in turn, affects critical parameters for biocontrol
agents spore germination and shelf-life.
Ultrastructural studies of mycoparasitism of T harzianum KMD on a soil-borne pathogen,
Rhizoctonia solani were investigated. The modes of antagonistic action by Trichoderma in
biological control have not been fully elucidated. However several mechanisms have been
described, such as mycoparasitism, antibiotics, production of inhibitors, which have been
identified and shown to suppress soil-borne pathogens. Mycoparasitic activities of T
harzianum KMD against R. solani were studied using in vitro bioassays and Scanning
electron microscopy (SEM). The fungal growth in dual cultures revealed that T harzianum
KMD made hyphal contact with the pathogen within four days of inoculation, leading to an
inhibition of pathogen growth. SEM observations showed that T harzianum KMD bound
firmly to R. solani hyphae by coiling around the hyphae. Penetration of the pathogens hyphae
occurred by the formation of hooks, haustoria and appressoria-like structures by T harzianum
KMD, followed by cell disruption. The pathogen's hyphae disintegrated and collapsed upon
contact with T harzianum KMD. It is hypothesized that the outcome of the interaction of
antagonist and pathogen was most likely determined by initial hyphal contact that triggered a
series of events in pathogen destruction.
An experimental trial was undertaken to evaluate various formulations if T harzianum KMD
and Gliocladium virens Strain MM1 for growth stimulation and biocontrol of R. solani and
Pythium sp. on a variety of crops under greenhouse conditions using three application
techniques at various dosages. Preparations of isolates of biocontrol agents T harzianum
KMD, G. virens MM1 and Bacillus subtilis Strain AW57 were evaluated for their efficacy in
enhancing growth and preventing damping-off caused by Pythium sp. and R. solani on a
variety of crops namely cabbage, cucumber, Namaqualand daisy and Eucalyptus. Percentage
survival and plot weights were measured after 3-4 weeks of growth. The preparations that
were used included chlamydospores of biocontrol fungi in milled oats, powders containing
conidia in an experimental compound, an oil base, and a commercial product. Formulations of
bacteria were prepared with and without Nutristart. The evaluation of three delivery methods
were used namely, a seed coating using an adhesive, Pelgel®, capping (a preparation is
capped on the surface and incorporated into planting media) and as a drench (preparation
drenched on seed at planting). Various dosage levels 0.25, 0.5, 1, 5 and 10g/1 of each
formulation was mixed with water and drenched on seed at planting.
Growth promotion of seedlings varied for the different formulations of different biocontrol
organism. Overall, plot weight was significantly increased on all crops tested. Plant growth of
seedlings was consistently increased by all conidial formulations of T. harzianum KMD and
G. virens MMI. The best application technique that effectively delivered the biocontrol
agents to the target was seed treatment followed by drenching and capping. Most formulations
significantly increased plot weight on all seedlings ranging from 2000-5000% when
compared to controls and percentage survival was comparable to the controls. In most
instances it was recorded that all biocontrol organisms effectively enhanced growth of
seedlings equally well irrespective of other main effects.
Most formulations of the different biocontrol organisms significantly reduced damping-off
caused by Pythium sp. on eucalyptus and Namaqualand daisy. Formulations of T. harzianum
KMD prepared with chlamydospores in milled oats and prepared with conidia effectively
reduced damping-off on eucalyptus and Namaqualand daisy by 8-31% when compared to the
controls. It was observed that biocontrol organisms T. harzianum KMD and G. virens MMI
effectively reduced damping-off better than B. subtilis AW57.
To effectively reduce damping-off caused by Pythium sp. seed treatment was the best
application technique to deliver the biocontrol agent to the target. Biocontrol of damping-off
caused by R. solani was achieved on all crops by all formulations of T. harzianum KMD, G.
virens MMl and B. subtilis AW57. Disease was reduced by 1000 fold with the application of
biocontrol organisms when compared to disease controls. Conidial formulations performed
better in reducing disease than formulations prepared with chlamydospores applied as a
drench or a seed treatment. In most instances the best dosage to apply formulations were
doses that ranged from l-5g/1 for both growth stimulation and biocontrol of soil-borne
pathogens. Severe stunting of seedlings occurred at high dosages of 109/I.
The compatibility of the biocontrol agent T harzianum KMD with selected fungicides were
determined on a variety of crops under greenhouse conditions. A commercial formulation of
T harzianum KMD was used for this investigation. An in vitro assay was used to determine
the sensitivity of T harzianum KMD to a range of rates of two fungicides, Benlate® and
Previcur®. Trichoderma harzianum KMD was found least sensitive to both fungicides after
15 days of incubation at 25°C. The compatible mutants resulted in a lack of sporulation even
when induced with UV light. Greenhouse trials were then carried out on cabbage, cucumber,
Namaqualand daisy, eucalyptus and tomato. It was confirmed that T harzianum KMD
achieved better growth and biocontrol activity against R. solani and Pythium sp. when applied
without fungicides to infested and non-infested composted pine bark. Trichoderma harzianum
KMD was only compatible to fungicides when applied as a seed treatment prior to planting.
As a disease integrated management programme, seed treatment application of T harzianum
KMD may be compatible with fungicides for control of damping-off of seedling diseases
caused by R. solani and Pythium sp.
The effect of environmental stress (oxidative injury, cold and drought) on the growth
enhancement of a variety of greenhouse crops by a commercial formulation of T harzianum
KMD was evaluated. In an absence of a disease colonization by T harzianum KMD on maize
and cucumber roots in rhizotron studies increased root area by 3104 mm(2) and 1787, 48 mm(2)
respectively. Oxidative stress was carried out by applying 0.05% NaOCl, to cabbage,
cucumber and tomato seeds. This stress did not reduce vigor of seedlings and hence the effect
of subsequent treatment with T harzianum KMD on stressed seeds was not determined.
Treatments of imbibed but unemerged seeds of cucumber, tomato and white grain maize in
cold temperatures (5-100C night/day) for varying periods reduced subsequent growth. Seeds
treated with cold stress and T harzianum KMD did not display any growth enhancement. On
cabbage, cucumber, tomato and white grain maize seeds sown in various media, which
induced various levels of drought and water logging conditions, were not enhanced when
seeds were coated with T harzianum KMD. Overall, T. harzianum KMD did not enhance
growth under stressed conditions of oxidative injury, cold and drought.
The results presented in this thesis shows that T harzianum KMD has potential against soilborne
pathogens namely Pythium sp. and R. solani under greenhouse conditions. Applying
conidial formulations of T harzianum KMD using seed treatment and applying it at the
correct dosage may increase the turnover of seedling production in nurseries. Trichoderma
harzianum KMD can replace toxic fungicides and. fumigants under greenhouse conditions.
More trials and research are needed on a wider variety of crops and diseases if growth
promotion and biological control of T harzianum KMD are to be fully exploited.
Description
Thesis (M.Sc.Agric.)-University of Natal, Pietermaritzburg, 2002.
Keywords
Seedlings--Diseases and pests., Seedlings--Diseases and pests--Control., Biological pest control agents., Phytopathogenic fungi., Trichoderma., Gliocladium., Theses--Plant pathology.