Thesis (Ph. D. Agriculture (Plant Production)) -- University of Limpopo, 2016. / Nemarioc-AL and Nemafric-BL phytonematicides, had been researched and developed
from indigenous plants at the University of Limpopo, Green Technologies Research
Centre, under the auspices of the Indigenous Cucurbitaceae Technologies (ICT)
Research Programme. After the international 2005 cut-off withdrawal date of the highly
effective methyl bromide nematicide from the agrochemical markets, management
options on nematode population densities shifted to more environment-friendly
alternatives. Nemarioc-AL and Nemafric-BL phytonematicides as environment-friendly
alternatives to synthetic chemical nematicides had been consistent in nematode
suppression under diverse conditions. In order to avoid challenges similar to those
experienced with the use of synthetic chemical nematicides, the South African Fertiliser,
Farm Feeds, Agricultural Remedies and Stock Remedies Act No. 36 of 1947 (amended)
require that the product to be used in agriculture must first be registered with the
National Department of Agriculture, Forestry and Fisheries, after extensive efficacy and
bioactivity tests. The information on bioactivity of the phytonematicides is also critical in
the effective application of the product for efficient management of nematodes.
Information on bioactivities of Nemarioc-AL and Nemafric-BL phytonematicides on
nematodes, plant and soil was not available. This study comprised eight objectives: (1)
to examine whether (i) increasing concentration of cucurbitacin A and B would have
impact on second-stage juvenile (J2) hatch of M. incognita, (ii) the Curve-fitting
Allelochemical Response Dosage (CARD) model would quantify the three phases of
density-dependent growth (DDG) patterns on J2 hatch when exposed to increasing
cucurbitacin concentrations, (iii) computed J2 hatch inhibition concentration (EHIC) and
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CARD-generated D-values would be statistically similar, (iv) the CARD model would
provide information on minimum inhibition concentration (MIC) and (v) J2 hatch
inhibition would be reversible when cucurbitacins were diluted, (2) to determine whether
(i) increasing concentration of Nemarioc-AL and Nemafric-BL phytonematicides would
have impact on J2 hatch of M. incognita, (ii) the CARD model would quantify the three
phases of DDG pattern on J2 hatch when compared to increasing phytonematicide
concentrations, (iii) comparison of computed EHIC and CARD-generated D-values
would be statistically comparable in magnitudes, (iv) the CARD model would provide
information on MIC and (v) J2 hatch inhibition would be reversible when
phytonematicides were diluted, (3) to establish whether (i) increasing concentration of
cucurbitacin A and B would have impact on M. incognita J2 immobility, (ii) the CARD
model would quantify the three phases of DDG pattern on J2 immobility when compared
to increasing cucurbitacin concentration, (iii) comparison of computed J2 immobility
concentration and CARD-generated D-values would be statistically comparable in
magnitudes, (iv) the CARD model would provide information on MIC and (v) juvenile
immobility would be reversible when cucurbitacins were diluted, (4) to test whether (i)
increasing concentration of Nemarioc-AL and Nemafric-BL phytonematicides would
have impact on M. incognita J2 immobility, (ii) the CARD model would quantify the three
phases of DDG pattern on J2 immobility when compared to increasing phytonematicide
concentrations, (iii) comparison of computed J2 immobility concentration and CARD
generated D-values would be statistically comparable in magnitudes, (iv) the CARD
model would provide information on MIC and (v) juvenile immobility would be reversible
when phytonematicides were diluted, (5) to determine whether (i) increasing
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concentration of cucurbitacin A and B would have impact on M. incognita J2 mortality,
(ii) the CARD model would quantify the three phases of DDG patterns on J2 mortality
when compared to increasing cucurbitacin concentration, (iii) comparison of computed
lethal concentration (LC) and CARD-generated D-values would be statistically
comparable in magnitudes and (iv) the CARD model would provide information on
minimum lethal concentration (MLC), (6) to investigate whether (i) increasing
concentration of Nemarioc-AL and Nemafric-BL phytonematicides would have impact
on M. incognita J2 mortality, (ii) the CARD model would quantify the three phases of
DDG pattern on J2 mortality when compared to increasing phytonematicide
concentrations, (iii) comparison of computed LC and CARD-generated D-values would
be statistically comparable in magnitudes and (iv) the CARD model would provide
information on MLC, (7) to test whether (i) increasing concentrations of Nemarioc-AL
and Nemafric-BL phytonematicides would impact on M. incognita J2 infectivity of
susceptible tomato plant, (ii) the CARD model would quantify the three phases of DDG
pattern (iii) generated inhibition concentration (IC) and CARD-generated D-values would
be statistically comparable in magnitudes and (iv) the CARD model would provide
information on MIC and (8) to determine whether nematodes can serve as bioindicators
of Nemarioc-AL and Nemafric-BL phytonematicides in tomato plant roots/fruits, soil
types and organic matter at different depths. To achieve these objectives, reliability of
measured variables was ensured by using statistical levels of significance (P ≤ 0.05)
and coefficient of determination (R2), with validity ensured by conducting three
independent experiments over time. In Objective 1, pure cucurbitacin A and B
concentration effects on J2 hatch were significant, with both exhibiting DDG patterns.
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The DDG patterns demonstrated that J2 hatch was inhibited at low pure cucurbitacin
concentrations and slightly stimulated at higher cucurbitacin concentrations. At 24-, 48-
and 72-h exposure periods, cucurbitacin A reduced J2 hatch by 40‒67, 34‒66 and
34‒45%, respectively, whereas cucurbitacin B reduced J2 hatch by 12‒57, 3‒36 and
9‒54%, respectively. CARD model quantified the concentration ranges of the two pure
cucurbitacins associated with the phases of DDG patterns. The J2 hatch was highly
sensitive to cucurbitacin B and highly tolerant to cucurbitacin A, as shown by
sensitivities values of 0‒2 and 5‒20 units, respectively. The CARD-generated MIC
values for cucurbitacin A and B were 1.75‒2.88 and 1.31‒1.88 µg.mL-1, respectively.
The conventionally generated J2 hatch inhibition concentrations were higher than
CARD-generated D-values at all exposure periods for both pure cucurbitacins. The J2
hatch inhibition effect was not reversible for both pure cucurbitacins. In Objective 2,
Nemarioc-AL and Nemafric-BL phytonematicide concentration effects on J2 hatch were
highly significant (P ≤ 0.01), with both exhibiting DDG patterns. The DDG patterns
demonstrated that J2 hatch inhibition increased with increase in phytonematicide
concentrations. Relative to water control, Nemarioc-AL phytonematicide significantly
reduced J2 hatch at 48-, 72-h and 7-d by 22‒92, 3‒79 and 1‒42%, respectively,
whereas Nemafric-BL phytonematicide reduced it by 41‒93, 1‒80 and 12‒84%,
respectively. The J2 hatch inhibition was highly sensitive to Nemarioc-AL and Nemafric
BL phytonematicides, with sensitivity of 0‒1 and 0‒4 units, respectively. The
conventionally generated J2 hatch inhibition concentrations at 50 and 100% were higher
than CARD-generated D-values for both phytonematicides. The J2 hatch inhibition
effect was not reversible for both phytonematicides. In Objective 3, pure cucurbitacin A
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and B concentration effects on J2 immobility were significant, with both exhibiting DDG
patterns. The J2 immobility over increasing concentrations of pure cucurbitacins had
DDG patterns which were similar for conventional method and those from CARD model.
The DDG patterns were characterised by stimulation of J2 immobility at low
concentrations, followed by saturation at higher concentrations. The CARD model could
not generate the D-values for comparison with JMC-values, but generated MIC-values
for cucurbitacin A and B which were 0.5‒0.6 and 0.5‒0.7 µg.mL-1, respectively. The J2
immobility was moderately sensitive to both cucurbitacins with sensitivity of 4 units and
the inhibition effect of the two pure cucubitacins was not reversible. In Objective 4,
Nemarioc-AL and Nemafric-BL phytonematicide concentration effects on J2 immobility
were highly significant (P ≤ 0.01), with both phytonematicides exhibiting DDG patterns.
The DDG pattern had stimulation, saturation and inhibition effects for Nemarioc-AL
phytonematicide, whereas for Nemafric-BL phytonematicide they had stimulation and
saturation effects on J2 immobility as concentrations increased. The MIC-values for
Nemarioc-AL and Nemafric-BL phytonematicides were 3.6‒115.2 and 0.1‒6.5%,
respectively. The CARD generated D-values were comparable with computed JMC
values for Nemafric-BL phytonematicide unlike for Nemarioc-AL phytonematicide. The
J2 immobility was highly sensitive to the two phytonematicides with sensitivity values of
0‒4 and 0‒2 units, respectively. The effects on J2 immobility of the two
phytonematicides were not reversible. In Objective 5, pure cucurbitacin A and B
concentration effects on J2 mortality were highly significant (P ≤ 0.01), with both
cucurbitacins exhibiting DDG patterns. The DDG pattern had stimulation, saturation and
slight inhibition effects for both cucurbitacin A and B as concentrations increased. The
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MIC-values for cucurbitacin A and B were 0.63 and 0.61 µg.mL-1, respectively. The
CARD-generated D-values were higher than the computed LC-values for both
cucurbitacin A and B, with J2 mortality being highly sensitive to cucurbitacin A and B,
with sensitivity of 4 units for both cucurbitacins. In Objective 6, Nemarioc-AL and
Nemafric-BL phytonematicide effects on J2 mortality were highly significant (P ≤ 0.01),
with both phytonematicides exhibiting DDG patterns. The DDG pattern had stimulation
effect at low phytonematicide concentrations and saturation effects at higher
concentrations for both relative impact and CARD-generated graphs of J2 exposed to
both phytonematicides. The MIC-values for Nemarioc-AL and Nemafric-BL
phytonematicides were 1.12 and 0.67%, respectively. The CARD-generated D-values
were higher than the computed LC-values for both phytonematicides and J2 mortalities
were highly sensitive to Nemarioc-AL and Nemafric-BL phytonematicides with sensitivity
value of 2 and 1 units, respectively. In Objective 7, Nemarioc-AL and Nemafric-BL
phytonematicide concentrations had a highly significant effect on infectivity of M.
incognita post-exposure on susceptible tomato seedlings. The relationship between
infectivity and increasing concentrations of the two phytonematicides exhibited DDG
patterns. The DDG patterns were characterised by stimulation effect at low Nemarioc
AL phytonematicide concentrations and saturation effects at higher phytonematicide
concentrations, whereas for Nemafric-BL phytonematicide slight inhibition, saturation
and stimulation effects were observed. The CARD-generated inhibition concentrations
for Nemarioc-AL phytonematicide were comparable with computed inhibition
concentrations, whereas for Nemafric-BL phytonematicides, the values were not
comparable. The MIC-values for Nemarioc-AL and Nemafric-BL phytonematicides were
xlvi
0.2 and 0.7%, respectively and J2 infectivity were highly sensitive to the two
phytonematicides, with sensitivity value of 2 and 0 units, respectively. In Objective 8, M.
incognita was an excellent bioindicator in response to the application of two
phytonematicides. The two phytonematicides significantly affected distribution of
population densities of M. incognita across the tested soil types, with Nemafric-BL
phytonematicide reducing population densities of M. incognita relative to Nemarioc-AL
phytonematicide. The active ingredient of Nemafric-BL phytonematicide, cucurbitacin B
tended to remain in the top layers of soil, where more roots accumulated, thereby
reducing a relatively higher population densities of M. incognita than did active
ingredient of Nemarioc-AL phytonematicide, cucurbitacin A which moved with water
beyond the effective root zone. Soil type alone and phytonematicide alone had no effect
on nematode numbers, whereas the interaction of soil type, phytonematicides and
depth, the nematode population densities were inversely proportional to soil depth. The
interaction of clay with any of the two phytonematicides, reduced M. incognita
population densities compared to sand and loam interactions. More than 62% tomato
root systems occurred in the top 0–25 cm depth. The interactions between organic
matter levels, phytonematicides and depth had no effect on the population densities of
M. incognita. The two phytonematicides were able to reduce nematode population
densities throughout the soil column in all four soil types and organic matter levels.
Cucurbitacin residues were not detected in all tomato fruit samples. In conclusion,
Nemarioc-AL and Nemafric-BL phytonematicides have bioactivities on J2 hatch, J2
immobility, J2 mortality and J2 infectivity. The CARD model quantified the three phases
of DDG patterns for most of the variables. Even though CARD-generated inhibition
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concentrations at 50 and 100% were not comparable with computed values for pure
cucurbitacins they were for most phytonematicide variables, the model was able to
generate excellent MIC-values for all variables. The inhibition effects of the two
phytonematicides were irreversible. The major findings of this study were that the two
phytonematicides exhibited DDG patterns for all variables tested and that the CARD
model could be adopted for the in vitro evaluation of phytonematicides. Meloidogyne
incognita was an excellent bioindicator on movement of two phytonematicides across
soil types and organic matter levels at different depths. Nemarioc-AL and Nemafric-BL
phytonematicides did not leave any cucurbitacin residues in tomato fruit. The
information on bioactivities of the two phytonematicides generated in this study provides
a much needed data for the registration of the products as required by the law.
Proposed future research area includes, microscopy study of molecular effects of the
phytonematicides on nematodes post-exposure. / National Research Foundation (NRF),
Flemish Interuniversity Council (VLIR) and Land Bank Chair-University of
Limpopo.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/1713 |
| Date | January 2016 |
| Creators | Dube, Zakheleni Palane |
| Contributors | Mashela, P. W., De Waele, D. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xlix, 267 leaves |
| Relation | Adobe Acrobat Reader |
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