More than 70 years ago, the Japanese philosopher Mokiji Okada, a proponent of natural farming, said that the difference between disease-resistant and susceptible varieties was the amount of nutrients in their bodies that allowed pathogens to thrive (Platani et al., 2023). This nutrient makes it easier for pathogens to grow, which makes it easy to get sick. On the other hand, if there is less nutrition conducive to the proliferation of pathogens, it is not easy to become infected (Li et al., 2021). Philosophers and social workers at that time did not know what these nutrients. Agronomists and plant nutritionists should know, but they could not know how to do studies at that time, so there was still no scientific mechanism at that time to figure it out. Only in the era of chemical fertilizers did nitrogen oversupply occur. At that time, at the general agronomic level, the technology of measuring the concentration of amino acids, amides, and nitrate ions had not been popularized, but the total nitrogen content of leaves was used to illustrate the phenomenon that excess nitrogen was conducive to disease (Nagajyothi et al., 2020).
The detailed mechanism is still unclear to the farmers of that era. Farmers only use the nitrogen content of leaves, or visually measure the deepness of leaf color to determine the amount of nitrogen nutrition, so as to make adjustments in subsequent topdressing or disease prevention measures. Joshi et al. (2022) showed a significant correlation between nitrogen fertilization and the severity of the rice blast. There are also great differences between varieties. As early as several decades ago, many studies confirmed that the amount of nitrogen fertilizer and the timing of fertilization have a great effect on rice leaf blast (dos Santos et al., 1986, Kurschner et al., 1992). However, these studies did not involve nitrate reductase or the NR1 gene. Perhaps because of limited research conditions, in developed areas with good research conditions, crop diseases have been successfully solved with pesticides. Very few people are doing research on this subject. Tomatoes, in particular, are now cultivated in protection facilities supported by high technology, with scientific water and fertilizer integration equipment and handy pesticides. Therefore, no one has paid much attention to the relationship between plant nutrition and disease. But grass-roots producers know that tomatoes cannot be over fertilized with nitrogen during vegetative growth, so they keep their tomato leaves more yellow-green than dark green. Once the leaf is dark green, it will be easy to get infected by pathogens. Tomatoes are some nutritious dietary source rich in antioxidant lycopene and vitamins (David, 2016). Tomato production is limited by several fungal pathogens, including Phytophthora infestans and Alternaria solani, which cause late and early stem and leaf wilt, respectively (Mengesha, 2017, Chasti et al., 2018). Up to 79% of tomato losses are due to the early blight (Singh et al. 2017, Gulzar et al. 2018). In particular, A. solani pathogens may overwinter in plant residues and persist in infection during subsequent roping seasons (Chaerani and Voorrips, 2006).
In the present study, all four tomato lines were infected with Alternaria solani, the early blight, and there was no specific pathogen inoculation. A. solani infection was marked by wilting on the leaves and concentric brown to black rings on the mature lower leaves. It then develops on the younger upper leaves (Roopa et al., 2014). Infected leaves turn pale yellow before wilting and shedding (Ogolla, 2021). On tomatoes, Phytophthora infestans does not only occur in the late growth period; in most cases, it starts early and occurs at all growth stages (Keskse, 2019). It causes plant death due to leaf and stem necrosis (Ogolla, 2021). On tomatoes, the onset of late blight is early, and the onset of early blight is relatively late. Because the appropriate temperature for the onset of late blight is 18°C–23°C and for the onset of early blight is 23°C–28°C, that is to say, late blight is the early onset at a low temperature, while early blight is the late onset at a high temperature. With the exception of Mosaic virus disease, early and late tomato blights are both associated with excessive use of nitrogen fertilizers. Nitrogen is an irreplaceable component of amino acids, proteins, nucleic acids, and chlorophyll (Kusano et al., 2011), which directly affects plant photosynthesis and metabolism (Muttucumaru et al., 2013, Zhang et al., 2020). This in turn affects plant growth, fruit yield, and quality (Yang et al. 2021). Many studies have shown that appropriate nitrogen application has positive effects on tomato yield and quality, including soluble sugars, vitamin C, sugar-acid ratio, and total phenol content (Cheng et al. 2021), while excessive nitrogen application has negative effects on tomato fruit quality. In particular, excessive nitrate concentration in leaves should not be ignored (Wang et al., 2015, Fan et al., 2022).
Many studies have confirmed that the up-regulated expression of the nitrate reductase gene enhances the activity of nitrate reductase, reduces the nitrate concentration in leaves, and improves the nitrogen absorption of plants (Du et al., 2012; Kyaing, 2011). Myoko F1 and its parents in this study were selected in organic fields with relatively low nitrogen supplies. Under the condition of limited nitrogen fertilization, nitrogen nutrition should be used as efficiently as possible, which is also one of the characteristics of organic varieties. However, nitrate reductase and nitrite reductase are not directly related to plant growth or yield. The conventional high-yielding variety Momotaro in the present experiment had low nitrate reductase activity, but the yield was not low, although slightly lower than Myoko F1, which was caused by the disease. There is no direct link between nitrate degradation capacity caused by increased nitrate reductase activity and plant growth (Hansch et al., 2001). Wang et al. (2003) once compared the resistance to tomato disease with the same variety treated with organic and chemical fertilizers. It was found that the nitrate reductase activity was higher, the concentrations of nitrate and amino acids were lower, and the ability to resist Phytophthora infestans was also stronger in tomato leaves with organic fertilizer.
In the present study, it was found that there was a significant positive correlation between nitrate reductase activity and disease resistance, and the correlation between NR1 gene expression and nitrate reductase activity was more significant (R2 = 0.9942). The resistant variety Myoko F1 is characterized by up-regulated expression of NR1 at the later growth stage, which reduces the concentration of intermediate nitrogen metabolites such as nitrate and free amino acids, thus enhancing disease resistance and delaying senescence. NR1 gene expression and average daily fruit yield were consistent with the modified Gaussian curve (an irregular bell curve) throughout the growth period. We used the modified Gaussian function model to simulate the trend of NR1 relative expression during the growth period, and the analysis parameters of the curve are listed in Table S2. Its equation is N = NM (1 + e(α(t-τ)2) ) + NB (1 + βt), where, τ is the number of days required for N to reach the maximum (NM + MB) and is also the peak of the curve.
The resistant variety Myoko F1 had a high τ value, the peak of the curve was shifted rightwards, and the aging of plant function was delayed. The average daily fruit yield of all tomato lines decreased in the later stage, but Myoko F1 still maintained a relatively high daily fruit yield, which could also be seen from the higher β value. The modified Gaussian equation used in this study can well simulate the variation trend of NR1 gene expression and daily fruit yield, especially since each term, each constant, and each coefficient in the equation each represent a physiological significance of the plant. The modified Gaussian curve integral (ꭍab ƒ (t) dt) and the median (ꭍab ƒ (t) dt)/(b - a) represent the meaning of the equation; in particular, the integral mean value is the average of the NR1 expression during the whole growth period. Both the integral value and the median value had a significant positive correlation with the disease resistance to leaf blight, and Myoko F1 was far greater than Momotaro and Myoko's parents. At the same time, the negative exponential curve equation was used to simulate the photosynthetic light response curve, and the characteristics of photosynthetic activity in each tomato line were well analyzed. Although there was no direct correlation between photosynthetic capacity and disease resistance at the seedling stage, photosynthetic activity in the later growth period was directly related to resistance to disease and fruit yield. The photosynthesis of the disease-resistant variety Myoko F1 was not higher than that of Momotaro at the early stage but significantly higher than that of Momotaro in the late growth period, which was consistent with disease resistance, nitrate reductase activity, and daily fruit yield. Therefore, it can be concluded that in this study, the activity of nitrate reductase and its gene expression were measured, and the mechanism of tomato resistance was accurately analyzed by mathematical model simulation. The research method and mathematical analysis we used can serve as a reference for similar research and have important implications for breeding disease-resistant varieties and guiding nitrogen management in tomato production.
In summary, the analysis of nitrate reductase genes and the mathematical models adopted indicate that the increase in nitrate reductase activity can promote nitrogen metabolism, reduce the accumulation of nitrate and amino acids in tomato leaves and fruits, and thus reduce the occurrence of tomato early leaf blight.