The goal of this thesis was to study factors related to the
development of Brassica juncea as a sustainable nematicide.
Brassica juncea is characterized by the glycoside (glucosinolate)
sinigrin. Various methods were developed for the determination of
sinigrin in Brassica juncea tissue extracts. Sinigrin
concentrations in plant tissues at various stages of growth were
monitored. Sinigrin enzymatically breaks down into allylisothiocyanate
(AITC). AITC is unstable in aqueous solution and
degradation was studied in water and in soil. Finally, the toxicity
of AITC against the root-lesion nematode (Pratylenchus penetrans)
A method was developed to extract sinigrin from whole Brassica
j uncea tissues. The optimal time of extraction wi th boiling
phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v)
solutions were both 25 minutes. Methanol/water extracted 13%
greater amount of sinigrin than phosphate buffer solution.
Degradation of sinigrin in boiling phosphate buffer solution
(0.13%/minute) was similar to the loss of sinigrin during the
extraction procedure. The loss of sinigrin from boiling
methanol/water was estimated to be O.Ol%/minute. Brassica juncea
extract clean up was accomplished by an ion-pair solid phase
extraction (SPE) method. The recovery of sinigrin was 92.6% and
coextractive impurities were not detected in the cleaned up
Several high performance liquid chromatography (HPLC) methods
were developed for the determination of sinigrin. All the developed
methods employed an isocratic mobile phase system wi th a low
concentration of phosphate buffer solution, ammonium acetate
solution or an ion-pair reagent solution. A step gradient system
was also developed. The method involved preconditioning the
analytical column with phosphate buffer solution and then switching
the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC
particle beam negative chemical ionization mass spectrometry (HPLCPB-
NCI-MS). Comparison of the mass spectra revealed the presence of
fragments arising from the ~hioglucose moiety and glucosinolate
Variation in the slnlgrin concentration within Brassica juncea
plants was studied (Domo and Cutlass cuItivars). The sinigrin
concentration in the top three leaves was studied during growth of
each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum
(1300~200~g/g) concentrations were observed at the third and
seventh week after planting, respectively. For Domo, the minimum
(190~70~g/g) and maximum (1100~400~g/g) concentrations were
observed at the fourth and eighth week after planting,
respectively. The highest sinigrin concentration was observed in
flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo
Physical properties of AITC were studied. The solubility of
AITC in water was determined to be approximately 1290~g/ml at 24°C.
An HPLC method was developed for the separation of degradation
compounds from aqueous AITC sample solutions. Some of the
degradation compounds identified have not been reported in the
literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide.
In water, AITC degradation to' diallyl-thiourea was favored at basic
pH (9.07) and degradation to diallyl-sulfide was favored at acidic
pH (4 . 97). It wap necessary to amend the aqueous AITC sample
solution with acetonitrile ?efore injection into the HPLC system.
The acetonitrile amendment considerably improved AITC recovery and
the reproducibility of the results.
The half-life of aqueous AITC degradation at room temperature
did not follow first-order kinetics. Beginning with a 1084~g/ml
solution, the half-life was 633 hours. Wi th an ini tial AITC
concentration of 335~g/ml the half-life was 865 hours. At 35°C the
half-life AITC was 76+4 hours essentially independent of the iiisolution
pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC
degradation was also studied in soil at 35°C; after 24 hours
approximately 75% of the initial AITC addition was unrecoverable by
The ECso of aqueous AITC against the root-lesion nematode
(Pratylenchus penetrans) was determined to be approximately 20~g/ml
at one hour exposure of the nematode to the test solution. The
toxicological study was also performed with a myrosinase treated
Brassica juncea extract. Myrosinase treatment of the Brassica
juncea extract gave nearly quantitative conversion of sinigrin into
AITC. The myrosinase treated extract was of the same efficacy as an
aqueous AITC solution of equivalent concentration.
The work of this thesis was focused upon understanding
parameters relevant to the development of Brassica juncea as a
sustainable nematicide. The broad range of experiments were
undertaken in support of a research priority at Agriculture and
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