Oat Grain Quality

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1		Michaela Havrlentová	+ 2018 word(s)	2018	2021-10-15 07:55:40	\|
2	update layout and reference	Rita Xu	Meta information modification	2018	2021-10-26 04:16:13	\|

This entry is adapted from the peer-reviewed paper 10.3390/microorganisms9102108

Oats (Avena sativa L.) belong to the main group of important cereal crops in Northern Europe and Northern America.

oat β-D-glucans fatty acids artificial inoculation Fusarium

1. Introduction

Oats (Avena sativa L.) belong to the main group of important cereal crops in Northern Europe and Northern America ^[1]. Oats are mainly used as feed for horses, but they also play an equally important role as an input material for food production and in generating pharmaceutical products and biomaterials ^[2]. Oats have an extensive cultivation history, playing a major role in human nutrition due to their starch and protein content, good amino acid balance ^[3], and their high fat content, with a relatively high ratio of unsaturated fatty acids ^[4]^[5]. Oat has a well-balanced nutritional composition. It is also a good source of total dietary fiber (TDF) and its soluble component β-D-glucans ^[3].

Oat fungal diseases are major limitations for this crop ^[6]. Alternaria and Cladosporium are general saprophytes or pathogens of an extensive variety of plants, and have been reported as the primary fungi in cereals worldwide ^[7]^[8]. Puccinina coronata f. sp. avenae causes crown rust disease in cultivated and wild oat (Avena spp.) ^[9]. Among the pathogenic fungi infecting oats, biotrophic pathogens, such as the powdery mildew agent Blumeria graminis f. sp. avenae, with very efficient mechanisms of spread, are the most difficult to control by means of crop management, such as rotation ^[6]. The main agent of root rot is Helminthosporium sativum ^[10]. Fusarium graminearum (FG), F. culmorum (FC), F. avenaceum, F. poae, and Microdochium nivale are the most common Fusarium head blight (FHB) pathogens ^[11]. In small grain cereals, FHB has emerged as a major problem in the Nordic European countries, and the impact of this disease in oats has been less investigated than in other cereals ^[12]. Taken together, these relatively few fungi can cause huge economic losses to agriculture, losses of food for consumption, and serious, often fatal diseases in humans and animals ^[13].

The Fusarium genus includes species that are widespread plant pathogens. These species cause diseases in different growing stages and parts of the plant, including the seedling blight, leaf spot, root rot, foot rot, and head blight. In cereals, FHB may be caused by at least 17 different Fusarium species ^[14]. Infection by Fusarium spp. is influenced by factors such as moisture and temperature, cultivar susceptibility, and cultivation practice ^[12]. The occurrence of the Fusarium species highly depends on the environment ^[15]. Due to changes in the climate and agricultural practices, changes in the order, as well the frequency of mycotoxins, are occurring ^[16]. FHB is mainly caused by FG and FC, which produce type B trichothecenes deoxynivalenol (DON) such as its acetylated derivatives, 3-acetyl-DON and 15-acetyl-DON, and nivalenol (NIV). Other species infecting cereal spikes include F. sporotrichioides, which produce type A trichothecenes T-2 toxin and HT-2 toxins, and F. poae ^[17]. The mycotoxin DON triggers potential cytotoxicity and genotoxicity in human peripheral blood lymphocytes via oxidative damage ^[18]. DON also causes abdominal distress, malaise, diarrhea, and emesis ^[19]. Trichothecenes are among one of the major groups of secondary metabolites produced by the species of the genus Fusarium and represent more than 200 compounds ^[20]. According to Gunnaiah and Kushalappa ^[21], the chemical defense against fungal pathogens, including DON produced by the Fusarium species, is associated with three main mechanisms of resistance: cell wall reinforcement through the deposition of lignin, the production of antimicrobial compounds, and the specific induction of defense signaling pathways. During pathogenic infection or biological elicitation, plant cells quickly respond by expressing genes encoding plant cell wall-associated non-enzymatic proteins. The cells then use the plant cell wall-associated non-enzymatic proteins to accomplish various defensive functions. These include the rapid reinforcement of cell wall strengthening against the pathogen penetration by insolubilization and the oxidative crosslinking of extensins and proline-rich proteins. In addition, the cells create a physical barrier against the pathogen invasion by clumping arabinogalactan proteins at the site of infection. The degradation of genetic materials of the pathogens by the binding of certain glycine-rich proteins with the RNA of the pathogens can be observed in the plant cell wall ^[22].

So far, nearly 40 identified metabolites have been reported to be associated with fatty acid metabolic pathways that may potentially affect cereal resistance against FG. Kachroo and Kachroo ^[23] summarized the importance of fatty acids and their derivatives in the defense of plants against pathogens. In addition to being essential for basal immunity and gene-mediated resistance in plants, fatty acids and their derivatives are also part of the induction of systemic acquired resistance and some of the breakdown products, such as oxylipins, and have a key role in the plant defense signaling pathway.

The oxylipin pathway is in plants a major defense signaling pathway. The oxidation of free polyunsaturated fatty acids, mainly linoleic and linolenic acid, presents the beginning of the biosynthesis of oxylipins. The action of lipoxygenases (LOX) is crucial in this step. The 9-LOXs and 13-LOXs are the main plant lipoxygenases, whereby the oxidation occurs at position 9 and 13 of the carbon chains, respectively. Two distinct biosynthetic pathways are induced. The 13-LOXs products are aimed at the formation of jasmonic acid and its derivatives. The 9-LOXs pathway leads to the production to lesser-known metabolites, which have important implications as defense factors in response to fungal attack ^[24]. The cultivation of oat varieties with high β-D-glucans content is another alternative to reduce the risk of grains contaminated by mycotoxins and thus to produce healthy and health promoting foods ^[25].

Oats (Avena sativa L.) have gained significant attention for their high content of dietary fiber, phytochemicals, and nutritional value. The consumption of oats has various health benefits, such as hypocholesterolemic and anticancerous properties ^[3].

2. Plant Material

Four different varieties of oats (Avena sativa L.) were used in the experiment. All oats were provided for research purposes by the breeder and curator of genetic resources of oats in the Slovak Republic, Peter Hozlár, Ph.D. (National Agricultural and Food Centre—Research Institute of Plant Production, Research and Breeding Station at Vígľaš-Pstruša, Detva). Ozon, Stoper, Vok, and SW Betania oats varieties were used. Ozon variety is of the hulled, yellow-grained type and originated from Germany. Ozon was registered in 2012. It is an early variety with big grains and a very good resistance to Pyrenophora leaf blotch and powdery mildew. Stoper is a hulled, yellow-grained type native to Poland, which was registered in 2003. It is a very early variety with a high-volume weight and very fine husk. Vok is a hulled variety of yellow-grained oats that originated from the Czech Republic. It was registered in 2002. Vok is a medium-late variety with fine husks and a very good resistance to crow rust, stem rust, and powdery mildew, as well as lodging. SW Betania is a spring variety of hulled, white-grained oats, and its country of origin is Sweden. The variety was registered in 2005. It is a medium-late variety with a very good resistance to crow rust, stem rust, and powdery mildew, BYDV, and lodging. Grains of oats were seeded in pots in three parallel replications. One replication was used as a control without the artificial inoculation Two replications were artificially inoculated. From each variety, 15 panicles were inoculated with FC and 15 inoculated with FG. In addition, 15 panicles of the control variant were collected. After harvest, mature grains were husked by hand and stored at −4 °C for further analysis.

3. Artificial Inoculation

Selected fungal isolates of FC and FG obtained from previous tests on cereal species were used to prepare the inoculum (data not shown). The Fusarium isolates were collected from commercial wheat fields in different regions of the Slovak Republic from naturally infected wheat spikes. The isolates were later preserved in the microorganism’s collection of the Research Institute of Plant Production (Piešťany, Slovak Republic). The fungal colonies of both FC and FG were cultivated on potato dextrose agar plates (PDA, Difco Lab., Detroit. MI, USA) for 21 days at 25 °C in the dark ^[26]. The inoculum was then prepared by covering the agar plates with sterile distilled water and soft scraping off the sporulated aerial mycelium. The hematocytometer was used to measure the concentration of inoculum which was regulated to 5 × 10⁶ conidia per ml. Approximately 1 mL of the conidial suspension was applied to each oat panicle.

For the artificial inoculation, the spraying method combined with polyethylene bag coverage was used ^[27]^[28]. The panicles were artificially inoculated at flowering using a spraying method with two separate inoculums of FC and FG. After inoculum application, the panicles were covered with polyethylene bags for 48 h.

4. Determination of Starch

The content of starch was determined using the Ewers polarimetric method (STN EN ISO 10520; 1997). The method is based on the partial hydrolysis of starch using hydrochloric acid. Then, the optical rotation of the obtained solution is measured. The optical rotation was measured using a sample cell of 200 mm optical path length at 20 °C. The final content of starch was recalculated for the dry weight of the sample.

5. Determination of TDF

The content of TDF was determined using the Total Dietary Fiber Assay procedure (Megazyme, Bray, Ireland). The method is based on the analytical methods published by Lee et al. ^[29] and Prosky et al. ^[30] (AOAC 991.43, AOAC 985.29, AACC 32-07, and AACC 32-05), and the AACC Method 32-21 and AACC method 32-06. TDF was determined in the dried samples in duplicate. The samples were incubated with heat-stable α-amylase at 100 °C to induce the hydrolysis and depolymerization of the starch. Next, protease was used at 60 °C to solubilize and depolymerize the proteins. The incubation with amyloglucosidase was used to hydrolyze the starch fragments to glucose. Samples were then treated with hot ethanol to remove the depolymerized protein and glucose from the starch and precipitate the soluble fiber. The precipitate was filtered and washed with ethanol and acetone. After drying, the precipitate was weighed. The nitrogen level was determined in one duplicate using the Dumas method (TruMac CNS Macro Analyzer, LECO Corp., St. Joseph, MI, USA). The second duplicate was used to determine the ash content. The final TDF content in the sample was then recalculated as the weight of the filtered and dried residue minus the weight of the protein and ash.

6. Determination of β-D-glucans

The total content of β-D-glucans in the grain was determined using the β-Glucan Assay Kit (Mixed Linkage) (Megazyme, Ireland). The streamlined method has been successfully evaluated by the AOAC (Method 995.16) and the AACC (Method 32-23), and published by McCleary and Codd ^[31]. Milled mature oat grains were suspended and hydrated in a sodium phosphate buffer (pH 6.5), incubated with purified lichenase, and filtered. Then, an aliquot of the filtrate was hydrolyzed with β-glucosidase. Oxidase/peroxidase reagent was used to examine the produced glucose. The content of the β-D-glucans was recalculated on the dry matter and determined as the mean of three replications as a percentage.

7. Determination of Lipids and Fatty Acids

The method by Soxhlet according to the standard (STN 461011-28) was used in duplicate to determine the total lipids content. To evaluate the fatty acids composition, methylesters were prepared according to Christoperson and Glass ^[32]. Next, they were analyzed by GC-MS (7890B GC system, 5977A MSD, Agilent Technologies, Santa Clara, CA, USA) using the capillary column HP-5MS ultra inert (30 m × 250 µm × 0.25 µm) and MS detector. The temperature gradient was used according to the following conditions: Column temperature program: initial temperature 150 °C (held for 4 min.), then increased to 230 °C (held for 5 min.) and finally increased to 280 °C (maintained 19 min.). The parameters of the capillary column inlet were as follows: temperature: 250 °C, pressure: 10.8 psi, total helium pressure: 169.32 mL/min., split ratio: 200:1. HP-5 ms ultra-inert column: dimensions 30 m × 250 μm × 0.25 μm, initial temperature: 150 °C, flow rate: 0.82746 mL/min., pressure: 10.8 psi, average value: 34 613 cm/s. The sample was injected at the volume 1 µL.

The MS parameters were as follows: MSD Transfer-line temperature: 280 °C, quadrupole temperature: 150 °C, ion source temperature: 230 °C, electron energy: 70 eV, record full mass spectra (SCAN type), scanning range: 50–550 m/z, scan speed: 1.562 (N = 2), gain factor: 1. Fatty acids were identified using the authentic standards of the C4–C24 fatty acids methyl esters mixture (Supelco, Bellefonte, PA, USA), and the mass spectrum of samples was compared with the spectrum in Library NIST2007 in ChemStation 10.1 (Agilent Technologies). All fatty acids were quantified as the area percentages of the fatty acids in the chromatogram.

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