Article pubs.acs.org/JAFC
Changes in the Concentration of Fumonisins in Forage Rice during the Growing Period, Differences among Cultivars and Sites, and Identification of the Causal Fungus Ryuichi Uegaki,*,†,⊥ Masanori Tohno,† Kohji Yamamura,§ and Takao Tsukiboshi† †
National Agriculture and Food Research Organization (NARO) Institute of Livestock and Grassland Science (NILGS), Senbonmatsu 768, Nasushiobara, Tochigi 329-2793, Japan § National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604 Japan ABSTRACT: To clarify the changes in rice fumonisin (FUM) concentrations, we conducted field cultivation of 10 forage rice cultivars and inoculation with fumonisin-producing fungal isolates. We cultivated 10 forage rice cultivars at the NARO Institute of Livestock and Grassland Science and one cultivar at two additional farmland sites in Japan in 2011 and 2012. Fusarium fujikuroi, which primarily infects plants shortly after heading, was inoculated on rice just after heading, and we sampled heads at the yellowripe and full-ripe stages to assess FUM concentrations. We found differences among cultivars in the FUM concentration and differences among the sites for the same cultivar, but no cultivar had high levels in leaves and stems. Fusarium fujikuroi was the main fumonisin producer. The FUM concentration in heads increases from 334.5 for FUMB1, m/z 706.6 > 336.5 for FUMB2; and confirmed ions, m/z 722.5 > 352.5 for FUMB1, m/z 706.6 > 318.4 for FUMB2. The efficiency of FUM recovery from rice flour with a known FUM concentration (100 ng/g) was 108.2% (FUMB1) and
days before heading and up to 12 times after heading in each year. Sampling involved harvesting the whole plant about 15 cm above the soil, and the harvested biomass was separated into two parts: stems and leaves, and the heads. Only the heads were sampled in 2012, because the concentration in the stem plus leaf component was quite low in 2011 (see the Results). Sampling was done in triplicate. These samples were used to determine the FUM concentration. Details of the analysis are provided in the section Quantitative Analysis of FUMs. Field Experiment 2: Differences in the FUM Concentration among Sites . We used the rice cultivar Yumeaoba in this experiment. Yumeaoba was cultivated at three sites in 2011 and 2012: the same experimental paddy field at NILGS that we used in field experiment 1, a farmer’s paddy field in Ohtawara, Tochigi Prefecture, and a farmer’s paddy field in Kumagaya, Saitama Prefecture. The sampling method was the same as in field experiment 1, but we did not use true replication at Ohtawara and Kumagaya; sampling was done in triplicate within the same field. Isolation of FUM-Producing Fungi. Isolation of FUMproducing fungi was carried out in 2011. To isolate the fungi, we placed 3 to 5 cm samples of the leaf and stem or head tissues on plates of potato dextrose agar medium (PDA, Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) containing 500 mg/L of streptomycin sulfate (Wako Pure Chemical Industries Co. Ltd., Osaka, Japan) and 500 mg/L of pentachloronitrobenzene (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The plates were incubated at 25 °C in the dark for 5 to 7 days, and the fungi present in the cultures were picked up and transferred into water agar medium. A single isolate was obtained from each sample, except for one sample that contained two or more Fusarium species. We then isolated individual fungal spores and incubated them on PDA for 5 to 7 days in the dark at 25 °C. The colonies on PDA were used in subsequent FUMproduction experiments and for identification to the species level. FUM-Production Experiment Using the Isolated Fungi. We incubated the isolates in the dark on 1 g of corn grits in 1 mL of sterilized water at 25 °C for 7 days. We used noninoculated corn grits as a control. We then added 10 mL of methanol/water solution (3:1 v/v) to the incubated corn grits, homogenized the sample by stirring using a glass rod, and then set it aside overnight. We used 5 mL of the supernatant for our quantitative analysis of the FUM concentration. If the FUM concentration was greater than 1 mg/kg of the corn grits, we defined the isolate as a FUM-producing fungus. Identification of Fusarium Species. We extracted total genomic DNA from the Fusarium isolates, as described previously.14 We amplified part of the gene for translation elongation factor 1-α (EF-1α) by means of the polymerase chain reaction (PCR) using the primer pair EF-1H/EF-2T, following the method of O’Donnell.15 We chose this gene because previous research15 showed that it was capable of distinguishing among Fusarium species and between these species and other organisms. Purified PCR products were sequenced with an ABI Prism 3100 automated sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The sequence data were compared with data in the DNA Data Bank of Japan (http://www.ddbj. nig.ac.jp/) by using the BLAST program. Greenhouse Experiment: Inoculation with FUMProducing Fungi. We used the cultivars Natsuaoba and Yumeaoba in this experiment. The greenhouse was kept at 25 3357
dx.doi.org/10.1021/jf405358h | J. Agric. Food Chem. 2014, 62, 3356−3362
Journal of Agricultural and Food Chemistry
Article
Figure 1. Changes in the fumonisin (FUM) concentration in the 10 cultivars during cultivation in field experiment 1 at the NILGS site. FUM concentrations represent the total (FUMB1 + FUMB2) content. Note that the y-axis scale differs among the graphs.
differences among the cultivars in the FUM concentration using Tukey’s HSD test. To compare cultivation sites, we analyzed the log-transformed averages using an unbalanced one-way ANOVA in which the numbers of replicates were 3, 1, and 1 for the NILGS, Ohtawara, and Kumagaya sites, respectively. When the ANOVA produced a significant result, we tested for differences among the sites using Tukey’s HSD test. To compare the FUM concentrations among the inoculation treatments, we tested for treatment × cultivar interactions using Tukey’s HSD test, because we suspected the existence of interactions. To compare the FUM concentrations among the inoculation treatments, we tested the mean for treatment × cultivar combinations using Tukey’s HSD test, because we suspected the existence of interactions. We calculated the mean using the log-transformed values of FUMB1 + FUMB2.
91.5% (FUMB2), with a relative standard deviation (n = 3) of 2.6% (FUMB1) and 4.5% (FUMB2). The calibration curve had good linearity (>0.999) in the range of 0 to 5000 ng/mL for FUMB1 and FUMB2. The limit of quantification (LOQ) was 1 μg/kg DM (FUMB1, FUMB2). Values below the LOQ were treated as not detected. The total FUM concentration was defined as the sum of the concentrations of FUMB1 and FUMB2. Statistical Analysis. To enhance the homoscedasticity that is required for performing ANOVA, we log-transformed the average FUM concentrations as ln(average +0.5w), where w is the maximum discrete width of the average. 17 To compare the cultivars, we analyzed the transformed averages using an unbalanced ANOVA in which the year (2011 and 2012) and the experimental plots (1, 2, and 3) were treated as blocks. When the ANOVA produced a significant result, we tested for 3358
dx.doi.org/10.1021/jf405358h | J. Agric. Food Chem. 2014, 62, 3356−3362
Journal of Agricultural and Food Chemistry
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Article
RESULTS Changes in the FUM Concentration during Cultivation and Differences among the Cultivars. Figure 1 summarizes the changes during the growing season in the FUM concentrations in the rice plants of the cultivars grown at NILGS in 2011 and 2012. Table 1 summarizes the mean FUM Table 1. Mean Fumonisin (FUM) Concentrations in the 10 Cultivars Cultivated in Experiment 1a mean (±SE)b
cultivar Fukuhibiki Bekoaoba Natsuaoba Hidekomochi Yumeaoba Hokuriku-193 Takanari Kusanohoshi Momiroman Mochidawara
6.150 5.922 5.873 5.201 4.920 4.102 4.014 3.480 2.741 1.976
(±0.529) (±0.778) (±0.529) (±0.778) (±0.529) (±0.529) (±0.778) (±0.529) (±0.529) (±0.778)
a ab ab abc abc abc abc bc c c
Figure 2. Differences in the FUM concentration between the two farm rice paddy sites. Values are the total FUM concentration (FUMB1 + FUMB2); the sampling date represents the number of days after heading. Upper (2): Ohtawara. Bottom (3): Kumagaya. Results for NILGS are shown in the graph for Yumeaoba in Figure 1. Note that the y-axis scale differs between the graphs.
a
Means represent log-transformed values, as described in the text. Means followed by different letters differ significantly (P < 0.05, Tukey’s HSD test). b
concentration in the heads of each cultivar. Fukuhibiki had a significantly higher FUM concentration than Kusanohoshi, Momiroman, and Mochidawara. Bekoaoba and Natsuaoba had a significantly higher FUM concentration than Momiroman and Mochidawara. The other cultivars did not differ significantly. The FUM concentrations in the stems and leaves of Hokuriku-193, Kusanohoshi, Fukuhibiki, and Momiroman were less than 10 μg/kg DM. The FUM concentration in stems and leaves of Hidekomochi at 40 days after heading were around 15 μg/kg DM (Figure 1). The FUM concentrations in stems and leaves of Natsuaoba were slightly higher than those in the other cultivars, reaching a maximum of 60 μg/kg DM. In the heads, the FUM concentrations in all cultivars 30 days after heading were less than 10 μg/kg DM. However, by 40 days after heading, the FUM concentration in Natsuaoba, Fukuhibiki, Bekoaoba, and Hidekomochi began to increase. The FUM concentration reached its maximum around 50 to 70 days after heading, except for an earlier range of dates for Hokuriku-193 (40 to 60 days). Although the maximum concentration differed among the cultivars, the trend did not differ. The FUM concentration was greater than 100 μg/kg in Fukuhibiki, Bekoaoba, Natsuaoba, and Yumeaoba, although there was high variation among the replicates. For example, the FUM concentration in Natsuaoba at 56 days after heading ranged from less than 1000 μg/kg DM to more than 3000 μg/ kg DM in 2012. Similarly, the FUM concentrations in Yumeaoba heads at this time ranged from less than 500 μg/ kg DM to more than 2000 μg/kg DM. Differences among the Sites in FUM Concentration. Figure 2 summarizes the FUM concentrations in Yumeaoba in the two paddy fields in Ohtawara and Kumagaya in 2011 and 2012. The result at NILGS is shown in the graph for Yumeaoba in Figure 1. Table 2 summarizes the statistical results. As in experiment 1, the maximum concentration was reached between 50 and 70 days after heading in both years and at both sites. In addition, the concentrations differed by more than 100% among the sites, and the NILGS site had a significantly higher mean concentration than the Kumagaya site. At the
Table 2. Differences in the Mean FUM Concentrations in Yumeaoba among the Three Field Sitesa mean (±SE)b
location NILGS Ohtawara Kumagaya
5.928 3.709 2.272
(±0.258) (±0.447) (±0.447)
a ab b
a
Values are the log-transformed means (see the text for details). Means followed by different letters differ significantly (P < 0.05, Tukey’s HSD test). b
Ohtawara site, the maximum FUM concentration was 193 μg/ kg DM. At the Kumagaya site, the maximum concentration was 113 μg/kg DM. The FUM concentration at Kumagaya was particularly low (
