Seed wintering and deterioration characteristics between weedy and cultivated rice
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Incidences of weedy rice continuously occurred in paddy fields because its shattering seeds were able to over-winter. In this research, the seed deterioration of weedy rice was investigated compared with cultivated rice, and the wintering characteristics of these two types of rice were investigated with the field wintering test, freezing resistance test, and accelerated aging test.
For the wintering test, the seeds of weedy rice were placed on the soil surface of a paddy with cultivated rice seeds during the 2008/2009 and 2009/2010 winter seasons from November to April. The viability of seeds after wintering was 4.3% for cultivated rice, but 92.7% for weedy rice in 2008/2009. In the second wintering test, the seeds were placed under flooded and dry paddy conditions. The seed viability of cultivated rice was 5% in dry paddy and 0.5% in flooded paddy, but weedy rice maintained a high viability during winter of 90% in the dry paddy and 61% in the flooded paddy. Following freezing treatment of the imbibed seeds, the seed viability was 78% for weedy rice and 16% for cultivated rice. The deterioration of seed tissue induced by freezing treatment was observed by the tetrazolium test. In an accelerated aging test at low temperature and soaking conditions, the seed viability of the weedy rice was 40% higher than the cultivated rice 90 days after treatment. During accelerated aging of seeds, the protein content remained higher in the weedy rice compared to the cultivated rice, and fat acidity remained lower in the weedy rice compared to the cultivated rice. Catalase and superoxide dismutase activity of the weedy rice was 4 times higher than that of the cultivated rice, and DPPH radical scavenging activity of the weedy rice was also much higher than for the cultivated rice.
In conclusion, the superior ability of seed wintering in weedy rice was based on freezing resistibility of embryo cellular tissue and higher antioxidant activity to protect seed deterioration during the winter season.
KeywordsWeedy rice Seed Wintering Deterioration Freezing resistance Antioxidant Accelerated aging
Traditional rice cultivation has been via transplanting of rice seedlings. However, due to the severe problems of agricultural water scarcity and farm labor shortage, direct-seeding methods have been practiced in many parts of the world (Cao et al. ; Savary et al. ; Tabbal et al. ; Tomita et al. ). In most countries, weedy rice infestation increased significantly after shifting from rice transplanting to direct seeding, and it was recognized as a noxious weed (Ottis et al. ; Suh ; Vaughan et al. ). Weedy rice infestations have been reported to have spread to 40–75% of the total area of rice cultivation in Europe, 40% in Brazil, 55% in Senegal, 80% in Cuba, and 60% in Costa Rica (Fogliatto et al. ).
The occurrence of weedy rice has very large effects on rice yield (Pantone and Baker ; Pantone et al. ). Weedy rice infestations are responsible for significant yield losses, which are particularly severe in short varieties and late planting cultivation (Fogliatto et al. ). Weedy rice is so productive that it can spread and cause major economic damage (Kane and Baack ), but it is impossible to control weedy rice during the rice cultivation period by herbicide because it belongs to the same species as the cultivated rice (Delouche et al. ; Suh ).
Weedy rice looks similar to cultivated rice in outward appearance, but has a high level of adapting ability to environmental stress in physiological traits. Being distinguishable by a red pericarp, it disperses immediately by shattering at maturity, guaranteeing its continuation (Chung ; Chung and Ahn ; Chung and Paek ; Ottis et al. ; Suh ). The shattering nature of weedy rice after ripening makes it difficult to be removed when cultivated rice is harvested. The occurrence of weedy rice becomes more severe the following year due to shattered seed present in the soil (Seong et al. ).
The reason of continuous incidences of weedy rice was that its shattering seeds were able to over-winter. In this respect, the deterioration of weedy rice seeds during winter might be different with cultivated rice in freezing resistance and antioxidant activities. There were no reports on the freezing resistance, but some reports of antioxidant activities related with seed deterioration in rice seeds (Bailly ). Generally, it has become increasingly accepted that damage resulting from reactive oxygen species (ROS) or oxidative stress plays a role in the seed aging process. ROS are highly reactive and may modify and inactivate proteins, lipids, DNA, and RNA and induce cellular dysfunctions. Plants require high contents of antioxidants such as ascorbate, α-tocopherol, glutathione, and phenolic compounds that can remove ROS as a defense mechanism (Schoner and Heinrich Krause ), and enhance the activities of antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) (Bowler et al. ). If balances are broken between ROS generation and removal in plant cells, oxidant losses occur along with damage to lipid peroxidation and cell membranes (Larson ).
In the present study, the wintering of shattered seeds of weedy rice on paddy fields was compared with cultivated rice, and seed deterioration and related characteristics concerning seed wintering are reported.
In the case of fat acidity, the initial values were 6.50 KOH mg/100 g in the weedy rice and 6.59 KOH mg/100 g in the cultivated rice, which were the same between the two varieties. Until 40 days after treatment, there were no significant changes of fat acidity, but there was a significant increase and difference between weedy rice and cultivated rice 50 days after treatment. Fat acidities of weedy rice and cultivated rice were 42.9 KOH mg/100 g and 23.9 KOH mg/100 g at day 70, and 44.1 KOH mg/100 g and 30.0 KOH mg/100 g at day 90, respectively.
In this study, the wintering rate of weedy rice seeds was about 90% on the dry paddy and 60–80% on the flooded paddy, which was much higher than that of cultivated rice (below 5%). This result was in agreement with several reports about the high germination percentage of weedy rice seeds placed in shallow depths or on the soil surface of paddy fields in the winter (Chung et al. ; Fogliatto et al. ; Ko ; Noldin et al. ; Seong et al. ). Therefore, if a seed of weedy rice spread, it could occur every year and exponentially increase in the rice field. Fogliatto et al. () highlighted the significant efficacy of winter flooding in reducing weedy rice infestations in paddy fields. This report is meaningful because the practice showed a significant reduction of weedy rice seed density compared to overwintering under dry conditions, and also winter flooding could favor the decay of seeds of several weeds including weedy rice (Nelms and Twedt ). However, results reported here indicated more than 60% of weedy rice seeds could overwinter under flood conditions. Therefore, winter flooding may not be completely effective but may be an accompanying method to control weedy rice in Korea.
Meteorological data on temperature and precipitation in Jeonju city during the seed wintering test in the 2008/2009 and 2009/2010 seasons
Days of subzero temperature
As shown in Figure 3, the freezing resistance of weedy rice seed was much higher than that of cultivated rice. Weedy rice surviving semi-wildly in rice fields is known to show a high level of adaptability to environmental stress (Chung ; Chung and Ahn ; Suh ). The weedy seeds that shatter on the surface of paddy fields in the autumn should have resistance to freezing in the winter to regenerate in the following germinating season, although there was no report on the freezing resistance of weedy rice seeds. In plants with freezing resistance, antifreeze proteins block the growth of ice crystals in the outside spaces of cells in the tissue, thus preventing cell damage derived from the repetition of freezing and thawing, and also have functions in transforming the generation of ice crystals by adhering to surfaces of ice (DeVries ; Jeong ; Yoo and Hwang ). In this way, antifreeze protein accumulates in the tissue in case of cold acclimation, and thus makes the plant not freeze to death even in freezing temperature. The tetrazolium test in Figure 4 showed that freezing damage of the cultivated rice began in overall tissue of the embryo and aleuronic layer of seed. But, in the weedy rice seeds after freezing treatment, those seed tissues were stained by tetrazolium solution to dark purple to show freezing resistance. Therefore, one hypothesis for a freeze resistance mechanism of weedy rice is that anti-freeze proteins accumulate in the embryo and aleuronic layer of the seed.
The accelerated aging test has been recognized as an accurate indicator of seed vigor and longevity, and may be generally induced by high temperature and high humidity (Hsua et al. ). The deterioration of seeds wintering on the field surface in temperate regions could be affected by high moisture content at low temperature as discussed earlier, thus accelerated aging can be induced by soaking at low temperature (4°C). The seeds deteriorated rapidly under this accelerated aging method, and there was a large difference in deterioration rate between weedy and cultivated rice. The viabilities of seeds after 90 days of accelerated aging were 4% in cultivated rice and 40% in weedy rice.
The deleterious effect of accelerated aging on the longevity of seeds was associated with the damage occurring to lipid, nucleic acid, and protein owing to oxidative stresses (Cakmak and Horst ; Fujikura and Karssen ; Hsua et al. ; Ito et al. ; McDonald ; Pilar ; Shewfelt and Purvis ; Vartapetian and Jackson ; Yan et al. ). In the accelerated aging test, the difference of protein contents and fat acidities in seeds between weedy and cultivated rice indicated that weedy rice had a higher protective system to aging stress than the cultivated rice.
Scandalios () reported that SOD and CAT were effective antioxidant enzymes for decreasing oxidant damage of cells by oxidative stress. The activity of SOD before and after the accelerated aging treatment for 90 days in weedy rice was much higher than in cultivated rice. However, the SOD activities of seeds rapidly increased and decreased with the peak occurring 10 days after treatment. SOD activity decreased if stresses persisted for a long time or excessive stresses were added (Kim and Lee ). CAT is considered a primary enzymatic defense against oxidative stress induced by senescence, chilling, dehydration, osmotic stress, wounding, paraquat, ozone and heavy metals (Kibinza et al. ). Shon et al. () reported that CAT activity had exhibited a decreasing tendency upon submerging stress in young rice seedlings, and appeared as the most sensitive enzyme to stress among antioxidant components, regardless of whether it was in seeds. Similarly, CAT activity was reduced after treatment in both the weedy and cultivated rice, where the activity of weedy rice persisted at a higher rate than the cultivated rice. Tanida () reported that CAT activity of the embryo in the seed soaked at low temperature had positive correlation with germination percentage after treatment. High SOD and CAT activities, as well as radial scavenging activity were considered to increase the vitality of seeds from excessive stress damage in soaking at low temperature.
In conclusion, the superior ability of seed wintering in weedy rice was based on the freezing resistance of embryo tissue and the ability of antioxidant enzymes and photochemicals to reduce seed deterioration.
Two japonica weedy rice, PBR and WD-3, and two Korean bred cultivars (japonica), Hopum and Ilpum, were used in this experiment. Prior to the laboratory-based experiment, they were regenerated in the experimental field in Chonbuk National University to obtain fresh seeds. The weedy rice were harvested at 30 days after heading, and the cultivated rice were harvested at 50 days after heading. Seeds were air dried until seed moisture content (SMC) became less than 14%. The SMC was measured by oven-drying method. The seeds were sealed in plastic bags, and stored at 4°C until use for about 7–8 months. When they were used, the SMCs were about 12% regardless of varieties.
Seed wintering test on the paddy field
In the first wintering experiment, Hopum, a bred cultivar, was cultivated in the dry paddy field where the weedy rice (WD-3) occurred at a rate of more than 500 plants per m2. After harvesting Hopum, naturally shattered weedy rice (WD-3) seeds and artificially shattered cultivated rice (Hopum) seeds were placed on the soil surface at 3 spots of the paddy field under wire net protection during winter from November 2008 to April 2009. Germination tests of seeds collected at 3 spots after wintering were conducted at 25°C for 14 days using 4 replicates of 100 seeds each in accordance with the International Rules for Seed Testing for Oryza sativa (ISTA ).
In the second wintering experiment, two weedy rice varieties (WD-3 and PBR) and two Korean cultivars (Hopum and Ilpum) were tested. The seeds of 4 varieties (200 g each) were kept on the paddy from November 2009 to April 2010. The paddy conditions were maintained as two types during winter. One was a dry paddy as was used for the first wintering test and the other was a flooded paddy simulated by plastic boxes (41.0 × 24.5 × 15.0 cm) filled with 10 cm depth of paddy soil (silty clay loam) and superabundant water. During wintering, the seeds (400 grains each from three replicates) were sampled 5 times for viability tests (ISTA ). The meteorological data on temperature, subzero temperature days, precipitation, and precipitation days during the wintering test for 2008/2009 and 2009/2010 is shown in Table 1.
Seed freezing test
To investigate the resistance of seeds under subzero temperature, the weedy (PBR, WD-3) and cultivated rice (Hopum, Ilpum) were assigned to freezing treatment as follows; the seeds were frozen at −10°C for 24 hours after imbibing at 4°C for 24 hours and then thawed at 4°C for 24 hours. The freezing method was repeated 1 to 3 times, and then the viability of the seeds was tested as stated above in seed wintering test. After freezing treatment, a tetrazolium test was also carried out to investigate the state of seed tissues. Soon after freezing treatment, seeds were kept at 25°C for 15–18 hours. Following that, seeds and embryos were cut vertically using a razor blade, placed in 1% tetrazolium solution (pH 6.5–7.5) at 30°C for 2 hours, and the staining patterns were observed under a dissecting microscope.
Seed viability and physiochemical test under accelerated aging treatment
To determine the deterioration of soaked seed at low temperature, the seeds of weedy rice (WD-3) and cultivated rice (Hopum) were kept soaking in distilled water at 4°C for 90 days as an accelerated aging treatment, and viability and physicochemical changes of seeds were observed at 10 day intervals. During accelerated aging treatment with three replicates, a 100 g of seeds per replicate was sampled at each sampling time. Among the sampled seeds, 400 grains per replicate was used for the viability test and the rest was ground with liquid nitrogen to powder by auto-mill (TK-AM5, Tokken Inc.) and then dried by freeze dryer (Clean Vac 8, BioTron). The powder was used to analyze protein content, fat acidity, superoxide dismutase (SOD) activity, catalase (CAT) activity and 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. A germination test was conducted as for the wintering test with four replicates.
T : amount of KOH (ml) used in sample
B : amount of KOH (ml) used in blank
W : moisture content of sample
8.33 : conversion factor according to sample amount and KOH concentration
For analysis of SOD and CAT activity, 1 g of seed powder was homogenized with 5 ml 0.1 M potassium phosphate buffer (pH 7.0) contains 1 mM EDTA and 1 mM DMSO. The homogenized samples were centrifuged at 30,000 × g for 20 min. at 4°C. The supernatant was stored at −80°C and used as a crude enzyme extract (Shon et al. ). For SOD activity test by the nitro blue tetrazolium (NBT) reduction method (Beyer ; Shon et al. ), 0.1 ml of 400 mM methionine, 0.1 ml of 2.5 mM NBT and 0.3 ml of 1 mM EDTA were vortexed in the glass test tube at 25°C for 3 min. for temperature balance. Then, 0, 20, 40, and 60 μl of enzyme extracts and 2.45, 2.43, 2.41 and 2.39 ml of 50 mM potassium phosphate buffer (pH 7.8) were added respectively to make final volume of 3 ml of enzyme and potassium phosphate buffer. 50 μl of 120 μM of riboflavin was added to each test tube. The test tubes were softly shaken and placed under fluorescent lamps for 15 min., and absorbance was measured at 560 nm by a spectrophotometer. One unit of SOD was defined as the amount of enzyme necessary to inhibit formation of blue formazan to reduce absorbance to 50% of blank’s.
For the evaluation of individual treatment means, all collected data from the complete randomized design with three replicates (four replicates in germination test, exceptively), subjected to analysis of variance using Statistical Analysis System (SAS 9.1).
This work was supported by a National Research Foundation of Korea Grant funded by the Korean Government (2011–0003554).
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.