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Soil physical–chemical properties and HMs concentrationsThe soil physical–chemical properties and HMs concentrations are summarized in Table 1. The soil mean pH value was 6.17 and ranged from 4.16 to 9.04 in different sites. The samples sites of level for pH ≤ 6.0(acidic soil), 6.0  7.5(alkaline soil) were 51.5%, 36.4% and 12.1%, respectively. The average content of TN, TP and TK were 1.33 g kg−1, 1.16 g kg−1 and 23.6 g kg−1, and ranged from 0.7 g kg−1 to 2.4 g kg−1, 0.22 g kg−1 to 20.8 g kg−1 and 10.3 g k g−1 to 28 g kg−1, respectively.The mean concentration of Cd, Hg, As, Pb, Cr, Cu, Ni and Zn were 0.221, 0.155, 9.76, 32.2, 91.9, 35.2, 37.1 and 108.8 mg kg−1. Except Cd, the average concentration of Hg, As, Pb, Cr, Cu, Ni and Zn exceeded 93.8%, 7.1%, 6.3%, 17.8%, 25.3%, 10.7%, and 32.4% the soil background values for Chengdu, respectively, which indicates that HMs are enriched to a certain extent in soil. The CV of the HMs in the agricultural soils increased in the order Ni(10.1%), Cr(10.9%), Pb(15.4%), As(21.4%), Cd(31.1%), Zn(57.8%), Hg(58.1%) and Cu(59.9%). The exceptionally high variability of Cu, Hg and Zn indicates that these metals differed greatly with respect to different sites, and the existence of abnormally high values is the main reason that the CV was high. It further indicates that Cu, Hg and Zn may be affected by external interference factors. The mean concentration of all HMs in soil were below the risk screening values for soil contamination (GB 15618-2018) (MEEC, 2018), however, the results showed that in 76, 1, 1, 6 and 2 of sample sites the level of Cd, Pb, Cr, Cu and Zn exceeded the risk screening values.Assessment of heavy metal(loid)s pollutionIndex of geo-accumulationThe index of geo-accumulation of HMs in the soil in the study area are shown in Fig. 2. In a descending order of magnitude of Igeo mean value, the eight elements were as follows: Hg(0.18)  > Zn(-0.22)  > Cu(-0.30)  > Cr(-0.36)  > Ni(-0.45)  > Pb(-0.51)  > As(-0.52)  > Cd(-0.82), indicating that the soil HMs Zn, Cu, Cr, Ni, Pb, As and Cd in the study area were generally in a no contamination according to the defined classes, while Hg was in uncontaminated to moderately contaminated.Figure 2Indexes of geo-accumulation, pollution indexes and potential ecological risk indexes of HMs in study aera. Circles at the top and bottom of box plots correspond to the maximum and minimum values, respectively. The square in the box plot is the average value. Horizontal lines at the top, middle, and bottom of the box plot correspond to 75% percentile, median, and 25% percentile, respectively.Full size imageThe Igeo values for Zn, Cu, Cr, Ni, Pb, As and Cd in more than 90% of samples were less than zero, only a few outliers in the soil were classified as moderately contaminated or worse. Which meaning point pollution at those sample sites. However, the Igeo values for Hg in 47.34%, 40.61%,10.66% and 1.40% of samples were belonged to  As  > Pb  > Ni  > Cu  > Hg  > Zn  > Cd. Cr, As and Pb were the largest contributors for both adults and children, accounting for 47.33% and 42.37%, 32.68% and 39.64%, 15.95% and 13.26%, respectively. Indicating that attention should be pain to the Cr, As and Pb elements due to their noncarcinogenic risk. Which was basically consistent with the research results of Bo et al.1 and Bao et al.32. Overall, The HMs of Cr, As and Pb were the main non-carcinogenic factors in soil in the study area, and the risk control of these elements should be strengthened.Carcinogenic risk assessmentThe CR of the HMs are shown in Table 5. Three exposure pathways were considered for Cd and Hg in our study. But As and Pb were considered carcinogenic by ingestion and inhalation, and Cr was considered carcinogenic through inhalation. The TCR mean values were 3.68 × 10−5 for adults and 6.27 × 10−5 for children, obviously were in the range 1 × 10−4 from 1 × 10−6, suggesting the TCR caused by HMs in the study area was acceptable on the whole, but it still exceeds the soil treatment threshold value 10−6. The CR average values through the three exposure pathways were CRing 3.65 × 10−5, CRinh 2.53 × 10−7 and CRderm 1.07 × 10−7 for adults, CRing 6.25 × 10−5, CRinh 1.12 × 10−7 and CRderm 4.50 × 10−8 for adults. Clearly the CRing was much larger than CRinh and CRderm for both adults and children, which indicates that oral ingestion is the major exposure pathway for CR. Which was consistent with the research results of Song et al.31 and Bo et al.1. For single HM, the CR value of Pb and Hg for adults were 2.72 × 10−5 and 8.6 × 10−6, and Pb, Hg and Cd for children were 4.63 × 10−5, 1.48 × 10−5 and 1.36 × 10−6, respectively, within acceptable criterion. Overall, the longterm health effects for adults and children are not serious at current single HM level.In this study, the total contents of HMs in the soils were used to assess health risk, the bioavailability of HMs were not considered, which may have caused the assessment results to be higher than the actual local situation1,31,42. In addition, because the parameters of health risk evaluation for children were set to be more sensitive than those for adults, the non-carcinogenic risk and carcinogenic risk for children were higher than those for adults2,43,44. However, those risks were at acceptable or negligible levels. Therefore, the study area is suitable for safe and clean production of hotbed chives. More

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The top-brand nonglutinous rice variety ‘Koshihikari’, which has a high palatability, is extremely susceptible to blast. Therefore, farmers apply fungicides over four times during the rice production season. As Koshihikari is sold by the Niigata brand, it has been traditionally viewed as having a high eating quality in Japan, and because of this, both farmers and consumers have requested that the multiline variety KO-BL be tested to determine if it is equivalent to Koshihikari before its introduction. Trials comparing Koshihikari and KO-BL were carried out in 2003 and 2004 in 594 and 622 fields covering 236 and 315 ha, respectively. These trials evaluated plant homogeneity, eating quality, and blast suppression using fewer fungicidal sprays. Following favorable results, in 2005, all Koshihikari were converted to KO-BL multiline variety covering an area of 94,000 ha. In addition, seed use and cultivation were restricted to the Niigata area to distinguish KO-BL from Koshihikari grown in other prefectures.Seed production and mixture processes are managed with precision by each prefectural official member (Fig. 1a). Original isogenic lines (ILs) were separately produced from the original stock in the original strain fields by the Niigata prefectural government. Using a precise mixture machine, the mixture of four ILs was then blended by weight in 2000 kg volumes, all multiplied by ten (giving a total volume of 20 t). Original production fields and commercial fields all used blended seeds that had been authorized by seed production farmers and commercial farmers in the 2003 and 2004 trials. Thus, it takes two years for seed production at the original strain field followed by the original production field for the preparation of commercial fields; thus, the seed mixture composition needs to be determined at least two years before introduction. Susceptible and resistant (effective) ILs were mixed at a ratio of 3:7 from 2005 to 2019 (Fig. 1b, Supplementary Table S1). Susceptible ILs, possessing Pia and Pii genes, were always mixed at a ratio of 1:2, but the composition of resistant ILs, containing Pita-2, Piz, Pib, Piz-t, and Pit genes, was changed every two to three years to avoid the breakdown of resistance6. These changes were determined by annually monitoring blast race distributions.Figure 1Representative seed production flow from original stock to commercial field and history of Koshihikari BL composition from 2005 to 2019 in Niigata Prefecture. (a) S1–S2, susceptible KO-BL; R1-R2, resistant KO-BL. Seeds obtained from original stock field at the Niigata Agricultural Research Institute. Seeds obtained from the original strain field and the original production field at both designated farmers’ fields. Commercial field (general farmers field) used for KO-BL production. Each field requires a year for seed production. (b) Pia and Pii, susceptible; Pita-2, Piz, Pib, Piz-t, and Pit, resistant. The proportion of susceptible KO-BLs and resistant KO-BLs was consistently 3:7 across years.Full size imageIn Niigata Prefecture, the predominant 5 blast races distributed from 1994 to 2004 were 001.0 (virulent to Koshihikari [Pik-s]), 003.0 (virulent to Pik-s and Pia), 005.0 (virulent to Pik-s and Pii), 007.0 (virulent to Pik-s, Pia, and Pii), and 037.1 (virulent to Pik-s, Pia, Pii, and Pik) (Fig. 2a, Supplementary Table S2). Because all the 5 races were virulent to Koshihikari, which had been widely cultivated in Niigata area during the years, there were no drastic race changes. In addition, genetic variations in blast resistance indicated that Koshihikari also harbored the Pish gene, and that the Pia, Pii, and Pik genes were also dominant in the Hokuriku region, including Niigata Prefecture21. Virulent blast races against the resistance genes Pish, Pia, Pii, Pi3, Pi5(t), Pik, Pik-s, and Pi19(t) were dominantly distributed in Niigata Prefecture22. These reports confirmed that Koshihikari had been susceptible to dominant blast races before KO-BL introduction.Figure 2Blast race change during the 1994–2019 period in Niigata Prefecture and the worst-case simulation of blast race dynamics in KO-BL during the 2005–2019 (years 1–15) period. Races and virulences are shown in Table 1. (a) A red line indicates the year (2005) when KO-BL was introduced. Races 007.0 and 037.1 became dominant after the introduction. (b) Actual races and their rates in 2004 and annual KO-BL compositions from 2005 to 2019 were set in the simulation. Parameters set in the simulation were as follows: maximum lesion number in a year, 10,000,000; weather condition, 10 (favorable); virulent mutation rate, 10–5; overwintering probability, 0.01; number of simulated years, 15; and number of simulation trials, 1000. The 1000 trial results for the lesion number increase in each race were averaged in each year and transformed into rates to show race dynamics. All simulation results are shown in Supplementary Table 6 in Supplementary information 2. The races 007.0 and 037.1 were also dominant until year 15 (correspond to 2019). Both actual and simulated race dynamics showed no outbreaks of the resistant composition of KO-BL.Full size imageIn the 2005 release year of KO-BL, the predominant blast races, 001.0 (virulent to Pik-s) and 003.0 (virulent to Pik-s and Pia), drastically decreased in distribution from 41.8% to 22.3% and 27.6% to 17.3%, respectively (Fig. 2a, Supplementary Table S2). Interestingly, races 001.0 and 003.0 rapidly decreased by 5.4% and 1.3% in 2006, respectively, even though especially Pia, which can be infected by the race 003.0, was used in the KO-BL composition. Because all ILs in the composition of KO-BL were resistant to race 001.0, and race 003.0 was only virulent to Pia, which made up 10% of the annual KO-BL composition (Table 1). In contrast, races 007.0 (virulent to Pik-s, Pia, and Pii) and 037.1 (virulent to Pik-s, Pia, Pii, and Pik) dominated from 2005 to 2019. The higher rate of race 007.0 detection was affected by 30% of the ILs composing the annual KO-BL were susceptible. The second highest rate of race 037.1 detection was affected by a number of factors: the high susceptibility of a minor cultivar that had Pii and Pik, the mosaic configuration of fields typical in Niigata, and the air-borne spread of race 037.1. To maintain consensus on KO-BL cultivation based on total blast suppression in Niigata, rarely detected races virulent to resistant ILs in commercial fields are strictly supervised by the prefectural government to avoid unnecessary confusion in Niigata residents.Table 1 Susceptible or resistant reaction of Koshihikari and KO-BL against blast races.Full size tableIn 2008, to mathematically support KO-BL composition changes, we developed a simulation software to estimate long-term blast race dynamics in multilines using a plant‒pathogen coevolution system23. The model calculated the persistence of resistant ILs to determine the optimal timing of changes to multiline variety compositions. To simulate race dynamics in KO-BL, we set five currently investigated races, 001.0 (virulent to Pik-s), 003.0 (virulent to Pik-s and Pia), 005.0 (virulent to Pik-s and Pii), 007.0 (virulent to Pik-s, Pia, and Pii), and 037.1 (virulent to Pik-s, Pia, Pii, and Pik), and their rates in 2004, as well as five emerging races, 043.0 (virulent to Pik-s, Pia, and Piz), 303.0 (virulent to Pik-s, Pia, and Pita-2), 003.2 (virulent to Pik-s, Pia, and Pib), 403.0 (virulent to Pik-s, Pia, and Piz-t), and 003.4 (virulent to Pik-s, Pia, and Pit) (see Fig. 2b, Supplementary Table S3) against five newly introduced respective resistant KO-BLs (see Fig. 1b, Supplementary Table S1) and the annual KO-BL compositions from 2005 to 2019. The worst case (severe epidemic) simulation result (Fig. 2b, Supplementary Tables S3 and S6) showed that race 007.0 (virulent to susceptible Pik-s, Pia and Pii) became the predominant race (77.4%), and race 037.1 (virulent to Pik-s, Pia, Pii, and Pik) remained at a low frequency (21.6%) until the fifteenth year (corresponding to 2019). In addition, super-race virulent to all KO-BLs did not emerge in this simulation. These suppression of outbreaks of newly emerged virulent races, including super-race on resistant KO-BL was apparently affected by 2–3 years of change in resistant KO-BL composition, and total suppression of blast occurrence decreasing the blast population. These results indicated that almost all the epidemics analyzed reflected actual race dynamics without affecting other minor races from other susceptible cultivars grown in Niigata, especially up to 2011. Thus, our decision support system provides an evaluation of KO-BL persistence and indicates the KO-BL composition changes needed for blast race population control in large areas. In addition, our simulation model may be useful for evaluating future KO-BL composition changes.Blast occurrence drastically decreased after 2005 (Fig. 3a, Supplementary Table S4). The average occurrence of leaf and panicle blast was 46.1% and 52.9% during the 1995–2004 period and 9.5% and 9.6% during the 2005–2019 period, respectively. This resulted in a blast suppression effect by 70% of the resistant composition in KO-BL. Current seed production fields are rarely contaminated with virulent races against resistant KO-BLs. This suggests that seed sanitation contributes to the suppression of virulent pathogen epidemics in multilines. In addition, induced resistance24,25 may have no effect on the practical use of multilines. Rice plants were found to induce a resistance response when inoculated with avirulent races of blast (those that stimulate protective responses to virulent race attacks). As the detection of several races in one area is rare and blast occurrence tends to be low, conditions that induce resistance in field situations do not occur. Fungicide applications to control blast in KO-BL and other minor cultivars decreased by approximately one-third during the 2005–2019 period compared with 2004 (Fig. 3b, Supplementary Table S5). Thus, the commercial scale use of crop diversity is clearly effective for the environmentally friendly control of airborne diseases.Figure 3Leaf and panicle blast occurrence from 1994 to 2019 and blast control area from 2004 to 2019 in Niigata Prefecture. (a) A red line indicates the year (2005) when KO-BL was introduced. (b) Gross fungicide spray area decreased by approximately one-third during the 2005–2019 period compared with 2004.Full size imageThe optimum long-term solution for pathogen population control using genetic diversity includes multilines. Blast occurrence in KO-BL introduced in Niigata, and the theoretical value of blast suppression in KO-BL tested at small scales, were reduced by approximately 10% compared to that of monoculture plots26,27,28. Thirty percent of susceptible ILs in KO-BL have the potential to improve compatible races with susceptible ILs and become predominant in large areas. This would contribute to the suppression of rapid increases in new virulent races emerging in the blast population. To maintain consensus on KO-BL cultivation based on total blast suppression in Niigata, rarely detected races virulent to resistant KO-BLs in commercial fields are strictly monitored by the prefectural government. Educating Niigata farmers ensures the long-term use of KO-BL. In fact, lower blast occurrence has been attributed to careful KO-BL cultivation and seed management.The implementation of genetically diversified homogeneous seed mixtures, rotations with resistant KO-BL, restricted KO-BL cultivation, and pathogen monitoring allowed rice quality to be maintained, diseases to be suppressed, and environmentally sound agriculture to be economically viable in Niigata. Collaboration among prefectural officers, farmers, and consumers in Niigata has resulted in safer rice production with good agricultural practices (GAPs) that meet sustainable development goals (SDGs). In addition, DNA tests differentiate KO-BL from the original Koshihikari for buyers, thereby prohibiting illegal distribution. Multiline varieties have been used in small areas in two different prefectures. For example, in Miyagi pref., Sasanishiki BL consisted of Pik, Pik-m, and Piz at ratios of 4:3:3 and 3:3:4 in 1995 and 1996, respectively. This composition was changed to Pik, Pik-m, Piz, and Piz-t at a ratio of 1:1:4:4 from 1997 to 2007 to prevent an increase in race 037.1 (virulent to the BL: Pik and Pik-m). In addition, an equal mixture of seven BLs (Pib, Pik, Pik-m, Piz, Piz-t, Pita, and Pita-2) was cultivated in 300 ha areas (maximum 4000 ha) from 2008 to 2014 without any outbreaks observed. In Toyama pref., the Koshihikari Toyama BL, which consists of resistant ILs, Pita-2, Pib, and Pik-p at a ratio of 4:4:2, was cultivated in an area of 300 ha and required a 50% reduction in chemical inputs from 2003 up to the present. Our model also calculated a greater than 50-year persistence in terms of the small area effect in both prefectural cases. This result depends on an insufficient pathogen population increase in virulent mutations against resistant ILs (data not shown). In this way, the practical use of a multiline provides control without the need for as much fungicide with or without a periodic change in IL composition. Our results demonstrate that the management of crop and pathogen coevolution can control diseases at large scales and, thereby, contribute to global food security. More

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