Influence of the fertilizers Slavol and Biohumus on potato leaf area and stomatal density

Different types of fertilizers, such as chemical fertilizers, organic fertilizers and natural fertilizers are available on the market. The type of fertilizer has a large impact on plant production quality. The aim of this study, which was conducted under field conditions in the village of Jurumleri near Skopje, North Macedonia, was to investigate the influence of the application of two different types of fertilizers on leaf area and stomatal density in potato. The influence of the microbiological fertilizer Slavol and the organic fertilizer Biohumus was investigated. Also, a control plot without any fertilizer was used. A statistical tool (ANOVA test) was used for data analyses. The results showed that a statistically highly significant difference in average leaf area was found between plants treated with Slavol and plants treated with Biohumus (p<0.001). Also, a statistically highly significant difference was detected in stomatal density between plants treated with Slavol and control plants (p<0.001). A very significant difference was noticed between plants treated with Slavol and plants treated with Biohumus (p<0.01). The microbiological fertilizer Slavol showed better results than Biohumus in our research.


Introduction
Potato (Solanum tuberosum L.) belongs to the Solanaceae family and it is a globally important crop plant producing high yields of nutritionally valuable food in the form of tubers. Potato tubers are quite rich in carbohydrates and leaves are rich in protein. Therefore, different plant parts can reliably be used as livestock feed (Yildirim et al., 2005).
The organic fertilizer Biohumus is used to increase soil fertility and yield of agricultural crops. Biohumus is the result of bio processing of livestock and poultry farming waste by the red Californian worm. In previous research, positive effects of Biohumus on plant production were confirmed (Madiyeva et al., 2018;Sonko et al., 2018;Aćimović et al., 2015). Research on bio-fertilizers is necessary for the understanding of their impact on human health, flora, fauna and soil.
Leaf area estimate is an important parameter in understanding photosynthesis, light interception, water and nutrient use, crop growth and yield potential (Smart, 1974;Williams, 1987). A nondestructive estimate of leaf area is imperative in many ecophysiological and agronomic studies. Plant stomata, the vital gate between the plant and atmosphere, may play a central role in plant/vegetation responses to environmental conditions, and they have been investigated from molecular and whole plant perspectives, as well as at ecosystem and global levels (Nilson and Assman, 2007). Many factors such as CO2 The aim of this research was to compare the effects of two different fertilizers (Slavol and Biohumus) on potato leaf area and stomatal density.
concentration, heat stress, salt stress, drought, precipitation change and plant density have effects on stomatal density (Xu and Zhou, 2008).

Material and Methods
The experimental plot was set up on an alluvial soil, in the village of Jurumleri near Skopje, Macedonia, during the cultivation seasons in 2019. Potato (Solanum tuberosum var. Safari) was used as research material. Safari is a medium-early variety, with a yellow skin color, a high yield potential, and a round-oval shape. The potato was cultivated from tubers and planted in the open field. During the growing season, the plants were treated with the organic fertilizer Biohumus (a liquid fertilizer made of Californian red worms) and the biofertilizer Slavol (which contains nitrogen fixing and phosphorus mineralizing bacteria). The fertilizers were foliarly applied at a concentration of 1 % solution.
The treatments were set in three replications in the following order: 1. Ø control -no use of organic fertilizer, 2. T-1 -foliar treatment with Biohumus, 3. T-2 -foliar treatment with Slavol. Each treatment was applied every 10 days. The treatments started when the plants were 30 % flowering (May 28, 2019 ), the second application of fertilizers was on June 6, 2019 and the third on June 16, 2019.
One month after the last application of fertilizers, leaf length and leaf width were determined by using 30 randomly selected leaf samples. Leaf samples from control plants were used for comparison. Potato leaf area was determined using the model of nondestructive estimation of potato leaf area by Mompie et al. (2014). Also, stomatal density (number per 1 mm 2 For data analyses, ANOVA (analysis of variance) test was used. The least significant differences were computed by the LSD test at the level of p<0.05, p<0.01 and p<0.001 of leaf area) on both the lower and upper leaf surface was assessed by preparation and microscope examination on nail polish replicas (Brewer, 1992). Average stomatal density was determined, based on 10 leaves per treatment using counts from three areas per leaf.

Results and discussion
Fertilizers are compounds given to plants to promote growth. They can be applied via the soil, for uptake by plant roots or by foliar spraying, for uptake through leaves. One of the methods for enhancing soil fertility and environmental protection in today's market conditions is the efficient use of agro-industrial wastes (manure, poultry litter, waste from meatpacking plants, sugar factories, canneries, sludge treatment plants, residues of crop production, vegetable growing, horticulture, etc.) (Chatellard, 2017).
The environmentally friendly fertilizer Biohumus has a large number of macro and microelements, as well as growth substances, vitamins, antibiotics, amino acids and useful microflora. On the other hand, the application of microbiological fertilizers in plant production can reduce the use of mineral fertilizers, which has an economic benefit, and ensures safe production. In that sense, Slavol as a microbiological fertilizer has shown a generally positive effect in plant production (Ðorđević et al., 2005;Jelačić et al., 2007;Masimović et al., 2012;Miskoska-Milevska et al., 2012;Najdenovska et al., 2012aNajdenovska et al., , 2012bNajdenovska et al., , 2013Miskoska-Milevska et al., 2018).
Leaf area measurements are required by plant physiologists to study primary production, by plant pathologists to be used as a measure of disease severity, and by ecologists who use leaf area relations in describing competition among different plant species (Korva and Forbes, 1995). Sometimes, it is necessary to determine leaf area without destroying the plants. Several non-destructive methods exist for leaf area determination. In this study, potato leaf area was estimated by the nondestructive model of Mompie et al. (2014).  According to the LSD-test, the difference in average leaf area between plants treated with Slavol and plants treated with Biohumus was statistically highly significant (p<0.001). No statistically significant difference in average leaf area was found between plants treated with Biohumus and control plants. The difference in average leaf area between plants treated with Slavol and control plants was not statically significant (Table 2). In contrast, the difference in average leaf area between strawberry plants treated with the microbiological fertilizer Slavol and control strawberry plants was statistically significant (Miskoska-Milevska et al. 2012). Also, Najdenovska et al. (2013) detected statistically significant difference (p<0.01) in average leaf area between treated broccoli plants and control broccoli plants. As reported by Miskoska-Milevska et al. (2018), statistically highly significant difference (p<0.001) was found in average length of cauliflower plants between plants foliarly treated with Slavol and control cauliflower plants.
Potato has amphistomatous leaves i.e. stomata are on the upper and lower epidermis. As expected, stomatal density on the lower epidermis was higher than on the upper epidermis. Stomatal densities on the upper and lower epidermis in potato plants treated with Slavol and Biohumus versus control plants are presented in Table 3. The average stomatal density on the lower epidermis, as well as total stomatal density were the highest in plants treated with Slavol, followed by plants treated with Biohumus (Table 3). LSD-test showed a statistically significant difference in average stomatal density on the lower epidermis between plants treated with Slavol and control plants (p<0.05). A statistically highly significant difference in average stomatal density on the lower epidermis was found between plants treated with Slavol and control plants (p<0.001). Also, a statistically very significant difference in average stomatal density on the lower epidermis was detected between plants treated with Slavol and plants treated with Biohumus (p<0.01) ( Table 4). Statistical analyses of data indicated a statistically highly significant difference between plants treated with Slavol and control plants regarding total stomatal density (p<0.001). A statistically very significant difference was found in total stomatal density between plants treated with Slavol and plants treated with Biohumus (p<0.01). No significant difference in total stomatal density was detected between plants treated with Biohumus and control plants (Table 5). The results of t-test indicated a statistically significant difference in total stomatal number between strawberry plants treated with Slavol and control strawberry plants (Miskoska-Milevska, 2012). Miskoska-Milevska (2018) found a statistically highly significant difference in total stomatal density between cauliflower plants treated with Slavol and control plants (p<0.001).

Conclusions
A statistically highly significant difference in average leaf area was found between plants treated with Slavol and plants treated with Biohumus (p<0.001). No significant difference was found in average leaf area between plants treated with Biohumus and control plants. The difference in average leaf area between plants treated with Slavol and control plants was not statically significant.
For stomatal density, a statistically very significant difference was found between plants treated with Slavol and plants treated with Biohumus (p<0.01). There was a statistically highly significant difference between plants treated with Slavol and control plants regarding total stomatal density (p<0.001). No significant difference was found in total stomatal density between plants treated with Biohumus and control plants. The microbiological fertilizer Slavol showed better results than Biohumus in our research.