2We had previously reported that manganese (Mn)-induced chlorosis is a serious problem 3 in ratoon sugarcane seedlings grown in acidic soils. To further monitor the progression of 4 chlorosis and elucidate the corresponding mechanism, both plant growth and nutrient 5 status of sugarcane plants and soils were investigated in the growth seasons of ratoon 6 cane seedlings in 2016 and 2018. The impacts of rainfall and ammonium on chlorosis 7 were also investigated hydroponically. The results showed that the chlorotic seedlings 8 could green in mid-summer; Mn content in the first expanded leaf decreased significantly, 9 whereas iron (Fe) content increased significantly during the progression of greening. The 10 leaf Mn content in the greened seedlings decreased by up to 78.1% when compared with 11 that in the initial chlorotic seedlings. The seedlings also showed a significant increase in 12 seedling height and weight of the expanded leaves, accompanied by a decrease in plant 13 Mn content during the progression of greening. Moreover, young seedlings with less Mn 14 content showed earlier greening than older seedlings with more Mn content. The 15 exchangeable ammonium content in the soils increased significantly during the 16 progression of greening, and the addition of 1 mM ammonium to the chlorotic seedlings 17 resulted in a decrease in leaf Mn content by up to 80%. Furthermore, leaf SPAD value 18 and Fe content increased by 2.0-fold and 1.4-fold, respectively, after rainwater was 19 applied to the chlorotic seedling. These results indicate Mn-induced chlorotic seedlings 20 can turn naturally green, and downregulation of plant Mn content, rainfall in summer, and 21 soil ammonium contribute to the greening of chlorotic seedlings.22
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