## 3 Results
### Chemical Analysis of Lotus Tures Damaged by Hirschmanniella Diversa  
We observed SEM-EDS images from sections exhibiting both black spots and healthy parts on the lotus tubers obtained from lotus cultivation field NI (Figure 2A,B). As a result, inorganic precipitation was observed on the black spots (Figure 2F) but not on the healthy parts (Figure 2C). Additionally, we confirmed that there were no nematodes around the black spots, which supports the previous observation that _H. diversa_ prefers to invade young lotus tubers [11]. Furthermore, the EDS elemental map-sum spectrum of the healthy parts of the lotus tuber revealed that most of the elements were C (64.6% (\(w/w\))) and O (32.7% (\(w/w\))), while Fe, P, and other elements were broadly distributed (Figure 2G,H). On the other hand, P and Fe were concentrated on the sections containing black spots (Figure 2D,E). Comparison of elemental map-sum spectra showed that on the surface of healthy lotus tubers, Fe and P were 0.24% (\(w/w\)) and 0.09% (\(w/w\)), respectively, while the values in sections containing black spots were higher, with 1.68% (\(w/w\)) Fe and 0.12% (\(w/w\)) P.  
The results of the SEM-EDS analysis indicated that the black spots on the lotus tuber surface were composed of phosphorus and iron. Therefore, we quantified the P and Fe concentrations of the black spots and healthy parts on the lotus surface from three different cultivars using ICP-OES (Figure 3). As a result, the P concentrations in the black spots ranged from 7.31-9.15 mg/g-dry, while those in the healthy parts ranged from 4.89-6.64 mg/g-dry (Figure 3A). Fe concentrations in the black spots and healthy parts were 0.95-4.09 mg/g-dry and 0.73-0.96 mg/g-dry, respectively (Figure 3B). The average P concentrations were significantly (\(p<0.05\)) different between black spots (8.34 \(\pm\) 0.94 mg/g-dry) and healthy parts (\(5.65\pm 0.87\) mg/g-dry). For the Fe concentrations, the mean value found in the black spots (2.35 \(\pm\) 1.60 mg/g-dry) was 2.7-fold higher than that found in the healthy parts (0.87 \(\pm\) 0.13 mg/g-dry), while the difference was not statistically significant (\(p\) = 0.25).  
Figure 1: The calibration curve of damage index values in relation to soil chemical characteristics of **(A)** Phosphate (P\({}_{2}\)O\({}_{5}\)), (**B**) cation exchange capacity (CEC), **(C)** electric conductivity (EC), (**D**) CaO, (**E**) total nitrogen (TN), (**F**) total carbon (TC), and (**G**) specific gravity (SG) obtained by linear regression analysis.  
Figure 3: Concentrations of phosphorus (P) and iron (Fe) in the black spots (Bs) and healthy parts (Ct) of the lotus tuber surface. “Koshuku”, “Matsukaze”, and “Power” are different lotus cultivars. (**A**) P concentration, (**B**) Fe concentration, and (**C**) mean values of Fe and P concentrations in the Bs and Ct. The statistical analysis was performed based on [PERSON]’s \(t\)-test.  
Figure 2: Photographic and scanning electron microscopy (SEM) images of sections of a lotus tuber. (**A**,**B**) are schematic images of collected sections of black spots and healthy parts of lotus tubers, respectively. (**C**–**E**) are sections of lotus tubers containing black spots. (**F**–**H**) are sections of healthy parts of lotus tubers. (**D**,**G**) are elemental maps of iron (Fe). (**E**,**H**) are elemental maps of phosphorus (P).