Metabolite Variation between Nematode and Bacterial Seed Galls in Comparison to Healthy Seeds of Ryegrass Using Direct Immersion Solid-Phase Microextraction (DI-SPME) Coupled with GC-MS.

Annual ryegrass toxicity (ARGT) is an often-fatal poisoning of livestock that consume annual ryegrass infected by the bacterium Rathayibacter toxicus. This bacterium is carried into the ryegrass by a nematode, Anguina funesta, and produces toxins within seed galls that develop during the flowering to seed maturity stages of the plant. The actual mechanism of biochemical transformation of healthy seeds to nematode and bacterial gall-infected seeds remains unclear and no clear-cut information is available on what type of volatile organic compounds accumulate in the respective galls. Therefore, to fill this research gap, the present study was designed to analyze the chemical differences among nematode galls (A. funesta), bacterial galls (R. toxicus) and healthy seeds of annual ryegrass (Lolium rigidum) by using direct immersion solid-phase microextraction (DI-SPME) coupled with gas chromatography−mass spectrometry (GC-MS). The method was optimized and validated by testing its linearity, sensitivity, and reproducibility. Fifty-seven compounds were identified from all three sources (nematode galls, bacterial galls and healthy seed), and 48 compounds were found to be present at significantly different (p < 0.05) levels in the three groups. Five volatile organic compounds (hexanedioic acid, bis(2-ethylhexyl) ester), (carbonic acid, but-2-yn-1-yl eicosyl ester), (fumaric acid, 2-ethylhexyl tridec-2-yn-1-yl ester), (oct-3-enoylamide, N-methyl-N-undecyl) and hexacosanoic acid are the most frequent indicators of R. toxicus bacterial infection in ryegrass, whereas the presence of 15-methylnonacosane, 13-methylheptacosane, ethyl hexacosyl ether, heptacosyl acetate and heptacosyl trifluoroacetate indicates A. funesta nematode infestation. Metabolites occurring in both bacterial and nematode galls included batilol (stearyl monoglyceride) and 9-octadecenoic acid (Z)-, tetradecyl ester. Among the chemical functional group, esters, fatty acids, and alcohols together contributed more than 70% in healthy seed, whereas this contribution was 61% and 58% in nematode and bacterial galls, respectively. This study demonstrated that DI-SPME is a valid technique to study differentially expressed metabolites in infected and healthy ryegrass seed and may help provide better understanding of the biochemical interactions between plant and pathogen to aid in management of ARGT.

Response of Aphid Parasitoids to Volatile Organic Compounds from Undamaged and Infested Brassica oleracea with Myzus persicae.

Headspace solid microextraction (HS-SPME) and GC-MS were used to investigate volatile organic compounds (VOCs) from cabbage plants infested and uninfested with green peach aphid Myzus persicae. The HS-SPME combined with GC-MS analysis of the volatiles described the differences between the infested and uninfested cabbage. Overall, 28 compounds were detected in infested and uninfested cabbage. Some VOCs released from infested cabbage were greater than uninfested plants and increased the quantity of the composition from infested plants. According to the peak area from the GC-MS analysis, the VOCs from infested cabbage consisted of propane, 2-methoxy, alpha- and beta pinene, myrcene, 1-hexanone, 5-methyl-1-phenyl-, limonene, decane, gamma-terpinen and heptane, 2,4,4-trimethyl. All these volatiles were higher in the infested cabbage compared with their peak area in the uninfested cabbage. The results of the study using a Y-shape olfactometer revealed that the VOCs produced by infested cabbage attracted Myzus persicae substantially more than uninfested plants or clean air. The percentage of aphid choice was 80% in favor of infested cabbage; 7% were attracted to the clean air choice and uninfested plants. A total of aphids 7% were attracted to clean air. Comparing between infested and uninfested cabbage plants, the aphid was attracted to 63% of the infested cabbage, versus 57% of the uninfested cabbage. The preferences of Aphidus colemani and Aphelinus abdominalis to the infested or uninfested plants with M. persicae and compared with clean air indicated that parasitoids could discriminate the infested cabbage. Both parasitoids significantly responded to the plant odor and were attracted to 86.6% of the infested cabbage plants.

Sequential Extraction of Proanthocyanidin Fractions from Ficus Species and Their Effects on Rumen Enzyme Activities In Vitro.

Three proanthocyanidin fractions per species were sequentially extracted by 50% (v/v) methanol−water, 70% (v/v) acetone−water, and distilled water from leaves of Ficus racemosa (fractions FR) and F. religiosa (fractions FRL) to yield fractions FR-50, FR-70, FR-DW, FRL-50, FRL-70, and FRL-DW. Fractions were examined for their molecular structure, effect on ruminal enzyme activities, and principal leaf protein (Rubisco) solubilization in vitro. All fractions except FRL-70 contained flavonoids including (+) catechin, (−) epicatechin, (+) gallocatechin, (−) epigallocatechin, and their -4-phloroglucinol adducts. The fractions FRL-50 and FRL-DW significantly (p < 0.05) inhibited the activity of ruminal glutamic oxaloacetic transaminase and glutamic pyruvic transaminase. All fractions inhibited glutamate dehydrogenase activity (p < 0.05) with increasing concentration, while protease activity decreased 15−18% with increasing concentrations. Fractions FRL-50 and FRL-DW completely inhibited the activity of cellulase enzymes. Solubilization of Rubisco was higher in F. religiosa (22.36 ± 1.24%) and F. racemosa (17.26 ± 0.61%) than that of wheat straw (WS) (8.95 ± 0.95%) and berseem hay (BH) (3.04 ± 0.08%). A significant (p < 0.05) increase in protein solubilization was observed when WS and BH were supplemented with FR and FRL leaves at different proportions. The efficiency of microbial protein was significantly (p < 0.05) greater in diets consisting of WS and BH with supplementation of F. racemosa leaves in comparison to those supplemented with F. religiosa leaves. The overall conclusion is that the fractions extracted from F. religiosa showed greater inhibitory effects on rumen enzymes and recorded higher protein solubilization in comparison to the F. racemosa. Thus, PAs from F. religiosa are potential candidates to manipulate rumen enzymes activities for efficient utilization of protein and fiber in ruminants.

Proanthocyanidins Modulate Rumen Enzyme Activities and Protein Utilization In Vitro.

This study investigated the principal leaf protein (rubisco) solubilization and in vitro ruminal enzyme activity in relation to the molecular structure of proanthocyanidins extracted from leaves of Anogeissus pendula and Eugenia jambolana. Six proanthocyanidin fractions were extracted by 50% (v/v) methanol−water followed by 70% (v/v) acetone−water and then distilled water from leaves of A. pendula (AP) and E. jambolana (EJ) to yield EJ−70, EJ−50, EJ−DW, AP−70, AP−50 and AP−DW. Fractions were examined for their molecular structure and their effects on sheep ruminal enzymes and solubilization of rubisco in vitro. All fractions significantly (p < 0.05) inhibited the activity of ruminal glutamic oxaloacetic transaminase and glutamic pyruvic transaminase. The fractions AP−50 and EJ−50 significantly inhibited the activity of the R-cellulase enzyme. Most of the fractions inhibited R-glutamate dehydrogenase activity (p < 0.05) by increasing its concentration, while protease activity decreased by up to 58% with increasing incubation time and concentration. The solubilization of rubisco was observed to be comparatively higher in A. pendula (16.60 ± 1.97%) and E. jambolana (15.03 ± 1.06%) than that of wheat straw (8.95 ± 0.95%) and berseem hay (3.04 ± 0.08%). A significant (p < 0.05) increase in protein solubilization was observed when wheat straw and berseem hay were supplemented with A. pendula and E. jambolana leaves at different proportions. The efficiency of microbial protein was significantly (p < 0.05) greater with the supplementation of leaves of A. pendula in comparison to E. jambolana. The overall conclusion is that the proanthocyanidins obtained from E. jambolana exhibited greater inhibitory activities on rumen enzymes, whereas A. pendula recorded higher protein solubilization. Thus, PAs from A. pendula and E. jambolana appear to have the potential to manipulate rumen enzyme activities for efficient utilization of protein and fiber in ruminants.

Evaluation of Aphicidal Effect of Essential Oils and Their Synergistic Effect against Myzus persicae (Sulzer) (Hemiptera: Aphididae).

The insecticidal activities of essential oils obtained from black pepper, eucalyptus, rosemary, and tea tree and their binary combinations were investigated against the green peach aphid, Myzus persicae (Aphididae: Hemiptera), under laboratory and glasshouse conditions. All the tested essential oils significantly reduced and controlled the green peach aphid population and caused higher mortality. In this study, black pepper and tea tree pure essential oils were found to be an effective insecticide, with 80% mortality when used through contact application. However, for combinations of essential oils from black pepper + tea tree (BT) and rosemary + tea tree (RT) tested as contact treatment, the mortality was 98.33%. The essential oil combinations exhibited synergistic and additive interactions for insecticidal activities. The combination of black pepper + tea tree, eucalyptus + tea tree (ET), and tea tree + rosemary showed enhanced activity, with synergy rates of 3.24, 2.65, and 2.74, respectively. Essential oils formulation was effective on the mortality of aphids. Fourier Transform Infrared Spectroscopy (FTIR) analysis showed that stability of a mixture of essential oils was not affected by store temperature (15, 25, and 35 °C) and the functional groups were not changed during storage. Based on our results, the essential oils can be used as a commercial insecticide against M. persicae.

Study on the Correlation between the Protein Profile of Lupin Milk and Its Cheese Production Compared with Cow's Milk.

Australian sweet lupin, the largest legume crop grown in Western Australia, is receiving global attention from the producers of new foods. To understand the effect of protein on cheese yield, lupin milk proteins were separated from the first, second, and third filtrations by cheesecloths. However, proteins from the first and second were analyzed using two-dimensional polyacrylamide gel electrophoresis; then, the isolated proteins associated with cheese production were identified. The research also focused on identifying the optimal method of cheese production based on the coagulation process, temperature, yield, and sensory evaluation. Lupin curds from the two cultivars, Mandelup and PBA Jurien, were produced using vinegar, lemon juice, starter culture, vegetable rennet enzyme as coagulant, as well as curd generated using starter culture and vegetable rennet enzyme. Cow's milk was used as a control. The results indicated that first-time filtration produced better extraction and higher yield of lupin proteins and cheese than the second filtration. A sensory analysis indicated that lupin cheese produced from PBA Jurien lupin milk using vinegar, 7.80% expressed as acetic acid, and ground in 45 °C water, was the most acceptable. The cheeses were examined for their protein, carbohydrates, fat, ash, and moisture contents. The concentration of protein was approximately 27.3% and 20.6%, respectively, in the cheese from PBA Jurien and Mandelup. These results suggest that lupin milk can adequately supply the proteins needed in human diets and, thus, could be used in the production of many existing products that require animal milk as an input.

Study on Effect of Extraction Techniques and Seed Coat on Proteomic Distribution and Cheese Production from Soybean Milk.

Soybean-based food products are a major source of protein. In the present study, proteins in soybean milk from seeds of the cultivar Bunya (Glycine max) were extracted using the cheesecloth and the centrifuge methods. The milk was produced through mechanical crushing of both whole and split seeds in water. Following separation by either the cheesecloth or centrifuge, proteins were isolated from the soybean milk by using thiourea/urea solubilisation and then separated them using two-dimensional polyacrylamide gel electrophoresis. The isolated proteins were identified by mass spectrometry. A total of 97 spots were identified including 49 that displayed different abundances. Of the two separation techniques, centrifuge separation gave higher protein extraction and more intense protein spots than cheesecloth separation. Eleven of the ß-subunits of ß-conglycinin, three of the α-subunits of ß-conglycinin, and four of the mutant glycinin showed different levels of abundances between separation techniques, which might be related to subsequent cheese quality. Notably, split-seed soybean milk has less allergenic proteins with four α-subunits of ß-conglycinin compared to whole-seed milk with eight of those proteins. The sensory evaluation showed that the cheese produced from split-soybean milk received higher consumer preferences compared to that of whole seed, which could be explained by their proteomic differences. The demonstrated reference map for whole and split-seed soybean milk could be further utilized in the research related to soybean cheesemaking.

Preliminary Study on the Differences in Hydrocarbons Between Phosphine-Susceptible and -Resistant Strains of Rhyzopertha dominica (Fabricius) and Tribolium castaneum (Herbst) Using Direct Immersion Solid-Phase Microextraction Coupled with GC-MS.

Phosphine resistance is a worldwide issue threatening the grain industry. The cuticles of insects are covered with a layer of lipids, which protect insect bodies from the harmful effects of pesticides. The main components of the cuticular lipids are hydrocarbon compounds. In this research, phosphine-resistant and -susceptible strains of two main stored-grain insects, T. castaneum and R. dominica, were tested to determine the possible role of their cuticular hydrocarbons in phosphine resistance. Direct immersion solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) was applied to extract and analyze the cuticular hydrocarbons. The results showed significant differences between the resistant and susceptible strains regarding the cuticular hydrocarbons that were investigated. The resistant insects of both species contained higher amounts than the susceptible insects for the majority of the hydrocarbons, sixteen from cuticular extraction and nineteen from the homogenized body extraction for T. castaneum and eighteen from cuticular extraction and twenty-one from the homogenized body extraction for R. dominica. 3-methylnonacosane and 2-methylheptacosane had the highest significant difference between the susceptible and resistant strains of T. castaneum from the cuticle and the homogenized body, respectively. Unknown5 from the cuticle and 3-methylhentriacontane from the homogenized body recorded the highest significant differences in R. dominica. The higher hydrocarbon content is a key factor in eliminating phosphine from entering resistant insect bodies, acting as a barrier between insects and the surrounding phosphine environment.

Proteomic Characterisation of Lupin (Lupinus angustifolius) Milk as Influenced by Extraction Techniques, Seed Coat and Cultivars.

Lupin seeds are rich in proteins and other essential ingredients that can help to improve human health. The protein contents in both whole and split seeds of two lupin cultivars (Mandleup and PBA Jurien) were used to produce the lupin milk using the cheesecloth and centrifuge method. Proteins were extracted from the lupin milk using thiourea/urea solubilization. The proteins were separated by a two-dimensional polyacrylamide gel electrophoresis and then identified with mass spectrometry. A total of 230 protein spots were identified, 60 of which showed differential abundances. The cheesecloth separation showed protein extractability much better than that of the centrifuge method for both the cultivars. The results from this study could offer guidance for future comparative analysis and identification of lupin milk protein and provide effective separation technique to determine specific proteins in the cheese-making process.

Physical, chemical and biological behaviour of fumigants on cottonseed.

Fumigation is required to protect cottonseed in storage and pre-shipment from insect pests and/or microorganisms. Fumigation of cottonseed with carbon disulphide (CS2), carbonyl sulphide (COS), ethanedinitrile (C2N2), ethyl formate (EF), methyl bromide (MB) and phosphine (PH3) showed that >85% of the fumigants disappeared within 5 h of exposure. COS maintained >20 mg L-1 for 24 h. After 1 day of aeration, 75%-85% of the absorbed COS and MB and 20%-40% of the absorbed CS2, EF and PH3 were released from treated cottonseed. The fumigant residues were reduced by 80% for COS, 50% for EF or MB and 25% for CS2 after 1 day of aeration. After 13 days of aeration, fumigant residues were reduced by 95% for MB, 65% for EF, 55% for CS2 and to natural levels in the COS residue. Carbon disulphide, COS, PH3, EF and C2N2 had no effect on the germination of cottonseed, but germination was reduced to 50% by MB. COS has potential as a fumigant for control of insect pests in cottonseed because it dissipates quickly and does not negatively impact germination. On the other hand, MB appears to strongly absorb and requires an extended period for residues to dissipate, and it negatively impacts germination.

Application of Direct Immersion Solid-Phase Microextraction (DI-SPME) for Understanding Biological Changes of Mediterranean Fruit Fly (Ceratitis capitata) During Mating Procedures.

Samples from three different mating stages (before, during and after mating) of the Mediterranean fruit fly Ceratitis capitata were used in this experiment. Samples obtained from whole insects were subjected to extraction with the two mixtures of solvents (acetonitrile/water (A) and methanol/acetonitrile/water (B)) and a comparative study of the extractions using the different solvents was performed. Direct immersion-solid phase microextraction (DI-SPME) was employed, followed by gas chromatographic-mass spectrometry analyses (GC/MS) for the collection, separation and identification of compounds. The method was validated by testing its sensitivity, linearity and reproducibility. The main compounds identified in the three different mating stages were ethyl glycolate, α-farnesene, decanoic acid octyl ester, 2,6,10,15-tetramethylheptadecane, 11-tricosene, 9,12-(Z,Z)-octadecadienoic acid, methyl stearate, 9-(Z)-tricosene, 9,11-didehydro-lumisterol acetate; 1,54-dibromotetrapentacontane, 9-(Z)-hexadecenoic acid hexadecyl ester, 9-(E)-octadecenoic acid and 9-(Z)-hexadecenoic acid octadecyl ester. The novel findings indicated that compound compositions were not significantly different before and during mating. However, new chemical compounds were generated after mating, such as 1-iodododecane, 9-(Z)-tricosene and 11,13-dimethyl-12-tetradecen-1-acetate which were extracted with both (A) and (B) and dodecanoic acid, (Z)-oleic acid, octadecanoic acid and hentriacontane which were extracted with (A) and ethyl glycolate, 9-hexadecenoic acid hexadecyl ester, palmitoleic acid and 9-(E)-octadecenoic acid, which were extracted with solvent (B). This study has demonstrated that DI-SPME is useful in quantitative insect metabolomics by determining changes in the metabolic compounds in response to mating periods. DI-SPME chemical extraction technology might offer analysis of metabolites that could potentially enhance our understanding on the evolution of the medfly.

Analysis of volatiles from stored wheat and Rhyzopertha dominica (F.) with solid phase microextraction-gas chromatography mass spectrometry.

BACKGROUND: Volatile organic compounds (VOCs) contribute significantly to food flavour and can be used as indicators of quality, age of storage, and hygiene condition of stored products. The VOCs in the headspace of three different samples - healthy wheat, Rhyzopertha dominica, and wheat with R. dominica - were analysed at 25°C by solid phase micro-extraction (SPME) coupled with gas chromatography-flame ionisation detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). All the experimental conditions were kept consistent except a polar column and a non-polar column were used to assess the differences in volatile fingerprints. RESULTS: A total of 114 volatiles were identified by both the polar and non-polar columns, of which 48 were specific to one of the three samples tested. The volatiles were mainly carbonyl chemical compounds such as aldehydes, ketones and alcohols. GC-MS results showed slightly more VOCs were identified from the polar column. The total number for the three samples was 43 from the polar column compared to 39 from the non-polar column. Conversely, 30 VOCs unique to a given sample were identified from the non-polar column compared to 18 from the polar column. CONCLUSION: The use of both polar and non-polar columns is essential to capture the full range of VOCs produced by the three specific sample types investigated. The data can form the basis of enquiry into the relationship between storage and grain quality, and insect infestation and grain quality by observing the impact that these circumstances have on the production of volatile organic compounds.

Ethyl Formate: A Potential Disinfestation Treatment for Eucalyptus Weevil (Gonipterus platensis) (Coleoptera: Curculionidae) in Apples.

Export of Pink Lady apples from Australia has been significantly affected by infestations of adult eucalyptus weevils (Gonipterus platensis Marelli). These weevils cling tenaciously to the pedicel of apple fruit when selecting overwintering sites. As a result, apples infested with live G. platensis adults lead to rejection for export. Since the Montreal Protocol restricted use of methyl bromide as postharvest treatment, it was necessary to consider alternative safer fumigants for disinfestation of eucalyptus weevil. Laboratory experiments were conducted using concentrations of 5, 10, 15, 20, 25, 30, 40, and 80 mg/liter of ethyl formate. Complete control (100% mortality) was achieved at 25-30 mg/liter of ethyl formate at 22-24°C for 24-h exposure without apples. However, with 90-95% of the volume full of apples, complete control was achieved at 40 mg/liter of ethyl formate at 22-24°C for 24-h exposure. No phytotoxicity was observed and after one day aeration, residue of ethyl formate declined to natural levels (0.05-0.2 mg/kg). Five ethyl formate field trials were conducted in cool storages (capacity from 250-900 tons) and 100% kill of eucalyptus weevils were achieved at 50-55 mg/liter at 7-10°C for 24 h. Ethyl formate has great potential for preshipment treatment of apples. Its use is considerably cheaper and safer than already existing fumigants like methyl bromide and phosphine.

Headspace Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry for Analysis of VOCs Produced by Phytophthora cinnamomi.

Volatile organic compounds (VOCs) from Phytophthora cinnamomi-infected lupin seedlings were collected by headspace solid-phase microextraction (HS-SPME). The sampling was done 28 to 44, 52 to 68, and 76 to 92 h after inoculation (HAI). The HS-SPME samples were analyzed by gas chromatography-flame ionization detector (GC-FID) to assess the differences in volatile compounds between the P. cinnamomi-infected lupin seedlings and the control. Three specific peaks were identified after 52 to 68 h with the infected lupin seedlings, at which time there were no visible aboveground symptoms of infection. Subsequently, the VOCs of five different substrates (V8A, PDA, lupin seedlings, soil, and soil + lupin seedlings) infected with P. cinnamomi and the corresponding controls were analyzed by gas chromatography-mass spectrometry (GC/MS). A total of 87 VOCs were identified. Of these, the five most abundant that were unique to all five inoculated substrates included: 4-ethyl-2-methoxyphenol, 4-ethylphenol, butyrolactone, phenylethyl alcohol, and 3-hydroxy-2-butanone. Therefore, these metabolites can be used as markers for the identification of P. cinnamomi in different growing environments. Some VOCs were specific to a particular substrate; for example, 2,4,6-rrimethyl-heptanes, dl-6-methyl-5-hepten-2-ol, dimethyl trisulfide, 6,10-dimethyl- 5,9-undecadien-2-ol, and 2-methoxy-4-vinylphenol were specific to P. cinnamomi + V8A; heptanes and 5-methyl-3-heptaneone were specific to P. cinnamomi + PDA; 3-methyl-1-butanol, ethyl acetate, 2-methyl-propanoic acid, ethyl ester, and ethyl ester 2-methyl-butanoic acid were specific to P. cinnamomi-inoculated lupin seedlings; and benzyl alcohol and 4-ethyl-1, 2-dimethoxybenzene were only detected in the headspace of inoculated soil + lupin seedlings. Results from this investigation have multiple impacts as the volatile organic profiles produced by the pathogen can be utilized as an early warning system to detect the pathogen from contaminated field soil samples. Data from this investigation have also illuminated potential metabolic pathways utilized by the oomycete during infection which may serve as potential targets for the development of specific control strategies.

Optimization of Headspace Solid-Phase Microextraction Conditions for the Identification of Phytophthora cinnamomi Rands.

A robust technique was developed to identify Phytophthora cinnamomi using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography (GC) coupled to a flame ionization detector (FID) for analyzing volatile organic compounds (VOCs). Six fiber types were evaluated and results indicated that the three-phase fiber 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) had the highest extraction efficiency for both polar and nonpolar GC columns. The maximum extraction efficiency (equilibrium absorption) was achieved 16 h after fiber exposure in the HS. Absorbed compounds on the fiber were completely desorbed in the GC injector after 5 min at 250°C. Compared with the nonpolar column, the polar column showed optimum separation of VOCs released from P. cinnamomi. Under the optimized HS-SPME and GC/FID conditions, lower detection limits for the four external standards was found to be between 1.57 to 27.36 ng/liter. Relative standard deviations <9.010% showed that the method is precise and reliable. The method also showed good linearity for the concentration range that was analyzed using four standards, with regression coefficients between 0.989 and 0.995, and the sensitivity of the method was 104 times greater than that of the conventional HS method. In this study, the VOC profiles of six Phytophthora spp. and one Pythium sp. were characterized by the optimized HS-SPME-GC method. The combination of the VOCs creates a unique pattern for each pathogen; the chromatograms of different isolates of P. cinnamomi were the same and the specific VOC pattern of P. cinnamomi remained consistently independent of the growth medium used. The chromatograms and morphological studies showed that P. cinnamomi released specific VOCs at different stages of colony development. Using the optimized HS-SPME GC method, identification of P. cinnamomi from 15 in vivo diseased soil samples was as high as 100%. Results from this study demonstrate the feasibility of this method for identifying P. cinnamomi and the potential use of this method for physiological studies on P. cinnamomi.

The Effect of Ozone Treatment on Metabolite Profile of Germinating Barley.

Ozone is widely used to control pests in grain and impacts seed germination, a crucial stage in crop establishment which involves metabolic alterations. In this study, dormancy was overcome through after-ripening (AR) in dry barley seed storage of more than 4 weeks; alternatively, a 15-min ozone treatment could break the dormancy of barley immediately after harvest, with accelerated germination efficiency remaining around 96% until 4 weeks. Headspace solid-phase microextraction (HS-SPME) and liquid absorption coupled with gas chromatography mass spectrometry (GC-MS) were utilized for metabolite profiling of 2-, 4- and 7-day germinating seeds. Metabolic changes during barley germination are reflected by time-dependent characteristics. Alcohols, fatty acids, and ketones were major contributors to time-driven changes during germination. In addition, greater fatty acids were released at the early germination stage when subjected to ozone treatment.

Ozone Efficiency on Two Coleopteran Insect Pests and Its Effect on Quality and Germination of Barley.

Ozone (O3) is a potential fumigant to control pests in stored grain since it can safely and rapidly auto-decompose without leaving residues. In this study, the efficacy of O3 on all life stages of Rhyzopertha dominica (Fabricius) and Tribolium castaneum (Herbst) in barley and the physiological effects on barley and its quality were investigated. Complete control of all life stages of pests was obtained at 700 ppm for 1440 min of ozone exposure without negatively impacting the contents of soluble protein, moisture content, seed colour, hardness, and the weight of thousand barley seeds. The eggs and pupae of these two insects were the more tolerant stages than their larvae and adults. Prolonged exposure times (40 to 1440 min) and mortality assessment intervals (1, 2, and 7 days) increased O3 efficacy due to the reaction characteristics and delayed toxicity. Aging barley seeds appeared to be more sensitive to prolonged ozone duration than new seeds. A total of 20 and 40 min could promote germination rate, and longer O3 exposure (1440 min) was unfavourable for germination and seedling growth. Thus, it is imperative to select an optimal O3 exposure time to transfer ozone into quality contributors of final products and achieve the desired functional outcomes.

Profiling Real-Time Aroma from Green Tea Infusion during Brewing.

Aroma substances are the most crucial criteria for the sensory evaluation of tea quality, and also key attractors influencing consumers to make the decision for purchasing tea. Understanding the aromatic properties of tea infusion during different brewing time is crucial to control the tea aromatic quality. Here, headspace and direct immersion solid-phase microextraction (HS-SPME and DI-SPME), coupled with GC-MS, were employed to investigate the impact of brewing time on the changes of the volatile features of green tea infusion. Esters, aldehydes, alcohols, fatty acids, and alkaloids were the predominant volatile groups from tea infusions. Two to three minutes was identified as the best duration for the tea brewing that can maximize the abundance of aromatic chemicals in the headspace emitted from the tea infusions. The variation of the key aromatic contributors between the tea infusion and the headspace over the infusion tended to equilibrate during the tea brewing process. This study provides a theory-based reference method by analyzing the real-time aromatic characteristics in green tea. The optimal time was determined for aromatic quality control, and the complementary relationship between the volatiles in the headspace and its counterpart, tea infusion, was primarily elucidated.

Tea-Soybean Intercropping Improves Tea Quality and Nutrition Uptake by Inducing Changes of Rhizosphere Bacterial Communities.

The positive aspects of the tea plant/legume intercropping system draw attention to the Chinese tea industry for its benefit for soil fertility improvement with low fertilizer input. However, limited information exists as to the roles of intercropped legumes in the rhizosphere microbiome and tea quality. Hereby, soybean was selected as the intercropped plant to investigate its effect on bacterial communities, nutrient competition, tea plant development, and tea quality. Our data showed that intercropped soybean boosted the uptake of nitrogen in tea plants and enhanced the growth of young tea shoots. Nutrient competition for phosphorus and potassium in soil existed between soybeans and tea plants. Moreover, tea/soybean intercropping improved tea quality, manifested by a significantly increased content of non-ester type catechins (C, EGC, EC), total catechins and theanine, and decreased content of ester type catechins (EGCG). Significant differences in rhizobacterial composition were also observed under different systems. At the genus level, the relative abundance of beneficial bacteria, such as Bradyrhizobium, Saccharimonadales and Mycobacterium, was significantly increased with the intercropping system, while the relative abundance of denitrifying bacteria, Pseudogulbenkiania, was markedly decreased. Correlation analysis showed that Pseudogulbenkiania, SBR1031, and Burkholderiaceae clustered together showing a similar correlation with soil physicochemical and tea quality characteristics; however, other differential bacteria showed the opposite pattern. In conclusion, tea/soybean intercropping improves tea quality and nutrition uptake by increasing the relative abundance of beneficial rhizosphere bacteria and decreasing denitrifying bacteria. This study strengthens our understanding of how intercropping system regulate the soil bacterial community to maintain the health of soils in tea plantations and provides the basis for replacing chemical fertilizers and improving the ecosystem in tea plantations.

Role of Lipids in Phosphine Resistant Stored-Grain Insect Pests Tribolium castaneum and Rhyzopertha dominica.

Insects rely on lipids as an energy source to perform various activities, such as growth, flight, diapause, and metamorphosis. This study evaluated the role of lipids in phosphine resistance by stored-grain insects. Phosphine resistant and susceptible strains of the two main stored-grain insects, Tribolium castaneum and Rhyzopertha dominica, were analyzed using liquid chromatography-mass spectroscopy (LC-MS) to determine their lipid contents. Phosphine resistant strains of both species had a higher amount of lipids than susceptible stains. Significant variance ratios between the resistant and susceptible strains of T. castaneum were observed for glycerolipids (1.13- to 53.10-fold) and phospholipids (1.05- to 20.00-fold). Significant variance ratios between the resistant and susceptible strains of R. dominica for glycerolipids were 1.04- to 31.50-fold and for phospholipids were 1.04- to 10.10-fold. Glycerolipids are reservoirs to face the long-term energy shortage. Phospholipids act as a barrier to isolate the cells from the surrounding environment and allow each cell to perform its specific function. Thus, lipids offer a consistent energy source for the resistant insect to survive under the stress of phosphine fumigation and provide a suitable environment to protect the mitochondria from phosphine. Hence, it was proposed through this study that the lipid content of phosphine-resistant and phosphine-susceptible strains of T. castaneum and R. dominica could play an important role in the resistance of phosphine.