Jenn-Wen Huang

Effect of ground Brassica seed meal on control of Rhizoctonia damping-off of cabbage

Ten cultivars of three Brassica species were compared for volatile compounds in hydrated ground seeds that were fungicidal to Rhizoctonia solani AG-4. Among the samples tested, the volatile substances in the ground seed of mustard (Brassica juncea cv. Bau Sin) showed the strongest fungicidal effect. Allyl isothiocyanate was one of the predominant fungicidal compounds detected in the ground seed of all Brassica species tested, and the amount was the highest in ground mustard seed meals. Among the nine carriers tested for seed coating with ground mustard seed meal, Biolan peat B3 was the most effective for the seed treatment. Cabbage seeds treated with a mixture of ground mustard seed meal and Biolan peat B3 (MBF mixture) at 2:3 ratio (w/w) resulted in a significant reduction in incidence of Rhizoctonia damping-off with no harmful effects to the germination of treated seeds. Effective control of Rhizoctonia damping-off of cabbage by the seed treatment with the MBF mixture was also obtained in a large-scale experiment conducted in a commercial automated environment-controlled greenhouse. This study suggests that the MBF mixture has potential for use in the commercial seed treatment.

Effects of ultrasonic and microwave pretreatments on lipid extraction of microalgae

AbstractnThe extraction of lipids from microalgal cells using ultrasonic and microwave pretreatments is mechanistically evaluated based on the distribution of cell fragments, the lipid content analysis, the scanning electron microscopic (SEM) observation of ruptured microalgal cells, and the analysis of fatty acids. The results indicate that microwave pretreatment extracts lipids more rapidly and efficiently as compared to ultrasonic pretreatment. The rupture of cells in the microwave process is due to the tremendous pressure caused by the rapid heating of the moisture inside the microalgal cells, whereas in the ultrasonic process the microalgal cells are ruptured by shock waves from cavitation bubbles outside the cells. The fatty acid composition of the respective lipids extracted via the two types of pretreatment did not vary significantly from one another. These results demonstrate that the microwave process is rapid and more effective than the ultrasonic process for lipid extraction from microalgae.

Direct determination of sinigrin in mustard seed without desulfatation by reversed-phase ion-pair liquid chromatography.

Reversed-phase ion-pair liquid chromatography has been investigated for directly analyzing sinigrin in mustard seed without desulfatation. After extraction by phosphate buffer (pH 7.0) from the grind-pastes of inactivated-myrosinase mustard seeds, sinigrin was first isolated through deproteinization and centrifugation, followed by filtration and injection into the chromatographic system. A reversed-phase C18 column was used to separate the sinigrin with an eluent of acetonitrile (ACN)-water (20:80) containing 0.02 M tetrabutylammonium (TBA) as the counter ion at pH 7.0. Detection was carried out with an UV detector operated at 227 nm. Factors affecting the chromatographic separation and quantitative determination, such as concentrations of TBA and ACN, and pH, were studied. The linear dynamic range is larger than three orders of magnitude and the detection limit is 0.045 mg/L. The RSD is around 3% and the recovery is 85% (3% RSD, n = 3).

Control, by Brassica seed pomace combined with Pseudomonas boreopolis, of damping-off of watermelon caused by Pythium sp.

A total of 57 isolates of fungi, bacteria, and actinomycetes were screened for their ability to degrade the glucosinolate sinigrin. Pseudomonas boreopolis was the most effective among 26 isolates having the capacity to degrade sinigrin. Treatment with ground Brassica seed pomace and Pseudomonas boreopolis resulted in the release of volatile compounds. Gas-chromatography analyses allowed to identify allyl isothiocyanate and 3-butenyl isothiocyanate as the major volatile compounds released from enzymatic hydrolysis of glucosinolates by Pseudomonas boreopolis. These compounds had strong fungicidal activity against Rhizoctonia solani AG-4, Sclerotium rolfsii, Sclerotinia sclerotiorum, Pythium aphanidermatum, and Phytophthora capsici as revealed by reduced mycelial growth. They were also responsible for reducing the survival of zoospores and oospores of P. aphanidermatum. A large-scale greenhouse trial showed that amendment of growth media (Bas Van Buuren medium No. 4, 7mL; soil, 3 g) with 1% (mass fraction) of the ground Brassica seed pomace and Pseudomonas boreopolis (1 × 108 to 1.8 × 108 cfu/mL) was effective in controlling damping-off of watermelon caused by Pythium sp., resulting in increased production of healthy, marketable seedlings.

Determination of Sinigrin in Vegetable Seeds by Online Microdialysis Sampling Coupled to Reverse-Phase Ion-Pair Liquid Chromatography

A hollow fiber microdialysis sampling coupled online to ion-pair liquid chromatography was investigated as an alternative to sample pretreatment for the direct determination of sinigrin in cruciferous vegetables without desulfation. After microdialysis, the dialysate was online injected into the chromatographic system to analyze the sinigrin with UV detection at 227 nm. Parameters affecting the microdialysis efficiency, such as flow rate, polarity modifier, pH in perfusion stream, pH, and salt added in sample solution, were studied. Through ion suppression in the donor phase and ion-pair formation in the acceptor phase, the microdialysis efficiency of sinigrin was enhanced. Experimental results revealed that the microdialysis of a sample solution (pH 2.0) using 0.1 M tetrabutylammonium (pH 12) as a perfusate at 10 microL/min flow rate maximized the extraction efficiency. Detection was linear in the concentration of 1.0-100 mg/L with a detection limit of 0.3 mg/L. Three seed samples were analyzed, with sinigrin at 49.8 (3.15% RSD), 20.0 (4.43% RSD), and 19.8 mg/g (4.22% RSD) for brussels, cauliflower, and cabbage, respectively. When 40 mg/g sinigrin was spiked in brussels seed powder, the recovery was 102.4% with 3.15% RSD (n = 3). The proposed method was proven to provide a very simple, rapid, and eco-friendly procedure to determine sinigrin in the seeds of cruciferous vegetables.

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Abstract Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.

Determination of alachlor and its metabolite 2,6-diethylaniline in microbial culture medium using online microdialysis enriched-sampling coupled to high-performance liquid chromatography.

In this study, a simple and novel microdialysis sampling technique incorporating hollow fiber liquid phase microextraction (HF-LPME) coupled online to high-performance liquid chromatography (HPLC) for the one-step sample pretreatment and direct determination of alachlor (2-chloro-2,6-diethyl-N -(methoxymethyl)acetanilide) and its metabolite 2,6-diethylaniline (2,6-DEA) in microbial culture medium has been developed. A reversed-phase C-18 column was utilized to separate alachlor and 2,6-DEA from other species using an acetonitrile/water mixture (1:1) containing 0.1 M phosphate buffer solution at pH 7.0 as the mobile phase. Detection was carried out with a UV detector operated at 210 nm. Parameters that influenced the enrichment efficiency of online HF-LPME sampling, including the length of the hollow fiber, the perfusion solvent and its flow rate, the pH, and the salt added in sample solution, as well as chromatographic conditions were thoroughly optimized. Under optimal conditions, excellent enrichment efficiency was achieved by the microdialysis of a sample solution (pH 7.0) using hexane as perfusate at the flow rate of 4 μL/min. Detection limits were 72 and 14 ng/mL for alachlor and 2,6-DEA, respectively. The enrichment factors were 403 and 386 (RSD < 5%) for alachlor and 2,6-DEA, respectively, when extraction was performed by using a 40 cm regenerated cellulose hollow fiber and hexane as perfusion solvent at the flow rate of 0.1 μL/min. The proposed method provides a sensitive, flexible, fast, and eco-friendly procedure to enrich and determine alachlor and its metabolite (2,6-DEA) in microbial culture medium.

Inhibition of Penicillium digitatum and Citrus Green Mold by VolatileCompounds Produced by Enterobacter cloacae

Penicillium digitatum causes green mold decay on citrus fruit, resulting in severe economic losses to citrus growers and packers worldwide. The present study is to evaluate the control of citrus green mold by volatiles produced by Enterobacter cloacae. An E. cloacae strain isolated from plant rhizospheres was able to produce three volatile organic compounds, which were identified as butyl acetate, phenylethyl alcohol, and 4,5-dimethyl-1-hexene by GC/MS chromatography. The volatile compounds produced by E. cloacae inhibited conidial germination and hyphal elongation of P. digitatum and reduced green mold severity. E. cloacae cultured at temperatures ranging from 16°C to 28°C, at pH values ≤6, or in a substrate carrier (sphagnum moss, vermiculite, or perlite) provided superior control against P. digitatum. A laboratory formulation using E. cloacae and perlite protected citrus fruit from green mold up to 22 days and its effectiveness outperformed fungicide application at room temperature (~25°C). The results implicate practical application of E. cloacae as a biofumigant for controlling citrus postharvest decay caused by P. digitatum. Significantly, the study provides a model for future research on how to formulate an effective biocontrol agent for disease management.