The Leaf Greenness Index SPAD and Selected Features of Potato Following an Application of Herbicides and Biostimulants

The objective of the research was to determine the effect of herbicides and herbicides combined with biostimulants on the leaf greenness index SPAD and selected potato characteristics, i.e. plant height, yield of large tubers and the vitamin C content. A three-year experiment was conducted to examine the following factors: factor I – three potato cultivars: Bartek, Gawin, Honorata; factor II – five methods of an application of herbicides and biostimulants: 1. control unit without chemical control, 2. the Harrier 295 ZC herbicide (linuron + clomazone), 3. the Harrier 295 ZC herbicide (linuron + clomazone) and the Kelpak SL biostimulant ( Ecklonia maxima ), 4. the Sencor 70 WG herbicide (metribuzin), 5. the Sencor 70 WG herbicide (metribuzin) and the Asahi SL biostimulant (sodium para-nitrophenol, sodium ortho-nitrophenol, sodium 5-nitroguaiacol). The cultivars investigated in the experiment significantly affected the leaf greenness index SPAD, plant height, yield of large tubers and the vitamin C content. The herbicides and biostimulants enhanced the values of the examined potato characteristics. Linear correlation coefficients confirm a strong, significant, positive correlation between the SPAD index and plant height, yield of large tubers and vitamin C content.


INTRODUCTION
Potato (Solanum tuberosum L.) belongs to one of three most important edible plants in the world, and is grown on all the continents [Birch et al. 2012]. Potato yields and quality are influenced by numerous agrotechnological, genetic and environmental factors [Escuredo et al. 2018, Trawczyński 2020, Wadas and Dziugieł 2020]. Appropriate tillage to control weeds is one of more important cultivation factors. Destruction of weeds by using a combination of mechanic and chemical practices contributes to increased tuber yields and improved tuber quality, as confirmed in the research by numerous authors [Ilić et al. 2016, Gugała et al. 2018, Barbaś and Sawicka 2020].
In recent years, there has been growing interest and application of various biostimulants in fields under crop plants, including potato, due to many sustainable agriculture-related benefits [Chehade et al. 2018, Nephali et al. 2020, Trawczyński 2020. Biostimulants stimulate sprouting, development of the rooting system and plant growth [Ertani et al. 2018]. Moreover, they increase the efficiency of chlorophyll synthesis, root development, nutrient uptake from the soil and nutrient metabolism, particularly when the environmental conditions are unfavourable for plant growth and development [Calvo et al. 2014, Popko et al. 2018. What is more, the products improve the plant resistance systems protecting them against abiotic stress and pathogens [Sharma et al. 2014].
In addition, they are safe for the environment and their application makes it possible to reduce the amount of chemicals utilised in agriculture and plant protection [Radkowski andRadkowska 2013, Du Jardin 2015].
The new regulation (EU) 2019/1009 defines plant biostimulants as follows: "EU fertilising product the function of which is to stimulate plant nutrition processes independently of the product's nutrient content with the sole aim of improving one or more of the following characteristics of the plant and/or the plant rhizosphere: (1) nutrient use efficiency, (2) tolerance resistance to (a) biotic stress, (3) quality characteristics, or (4) availability of confined nutrients in the soil or rhizosphere" [Regulation (EU) 2019]. The biostimulant product number and market are increasing year by year. In 2020, the list of growth stimulants in Poland included 64 products [Ministry of Agriculture and Rural Development 2021. Thus, the increasing range of biostimulants introduced into the market and their application in combination with other products prompt an assessment of a wide range of characteristics (physiological, morphological, chemical) of cultivated potato varieties. Potato is one of the most efficient plant species converting the solar energy into human food, both in qualitative and quantitative terms. The chlorophyll content in potato leaves is closely related to the potato content of nitrogen [Ramirez et al. 2014, Gaurav et al. 2019]. Vos and Bom [1993] demonstrated a very strong correlation (r = 0.97) between readings from the SPAD-502 measuring device manufactured by Minolta Camera as well as the nitrogen and chlorophyll concentrations in potato leaf laminae. Moreover, they found that the SPAD readings were closely correlated with the analytical measurements of chlorophyll contents, which ensures a good estimation of the leaf content of these components.
The measurement of chlorophyll SPAD with a measuring device is predominantly used to determine the nitrogen status in crop plants. , who applied black textile mulch while growing potatoes, found a strong correlation (r = 0.6572) between the SPAD values and tuber yield. The authors suggest that, based on the SPAD values, it is possible to predict the potato tuber yield levels as well as yields of its components. Good development of plants is indicative of their good nutrition, normal course of their life processes and, thus, optimum yielding and nutrient accumulation. The purpose of the work was to assess the effect of herbicides and herbicides combined with biostimulants on the leaf greenness index SPAD, tuber yield and the vitamin C content in tubers.

Field experiment and agrotechnological treatments
The field experiment was carried out for three years, from 2012 to 2014, on a farm of the multibranch company Soleks located in the District of Siedlce, east-central Poland. It was established in a complete block design with a split-plot arrangement, with three replicates. Two factors were examined in the experiment: • factor I -three medium-early During the growing season, the Colorado potato beetle was controlled with the following insecticides: Apacz 50 WG (clothianidin) and Fastac 100 EC (alpha, cypermethrin); the fungicides used against potato blight were as follows: Ridomil Gold MZ 68 WG (metalaxyl-M+mancozeb) and Altima 500 SC (fluazinam).

Leaf greenness index SPAD
The assessment of leaf greenness SPAD (Soil Plant Analysis Development) was conducted using a SPAD-502Plus measuring device (Konica Minolta, INC, Japan). The SPAD-502Plus chlorophyll meter is a small, handy, non-destructive device the readings of which are expressed in nonmetric SPAD units ranging from 0 to 200 [Chlorophyll meter SPAD 502Plus/502DL Plus]. It measures the light absorption by leaves at the wavelengths of 650 and 940 nm. The measurements were taken twice at 10-day intervals, beginning at the stage of full flowering (BBCH 67-68) and after chemical control treatments with herbicides and biostimulants Adamczewski and Matysiak [2011] ( Table 2).
Ten individual readings were taken in the second row of each plot starting with the third plant (on a second leaf from the top of the plant). The SPAD readings were taken in the morning between ten and noon on the same plants. Next, the values were recorded and averaged.

Plant height, tuber yield and determination of the vitamin C content
When the potato plants were fully developed, that is at the flowering stage (BBCH 67-68), 10 plants in each plot were analysed at three replicates. Each year, prior to harvest, tubers of randomly selected ten plants were dug out in each unit to determine the yield structure based on the following size fractions: 35 or less, 36-50, 51-60 and 60 mm or more. Total tuber yield comprised the weight of tubers manually harvested from each plot and the weight The weather data are presented in Table 3. On the basis of Sielianinov's hydrothermal coefficient, the year 2012 was dry, 2013 was optimum and 2014 was relatively dry [Skowera et al. 2014].
In 2012, the average air temperature in April-September was 15.4 °C and was by 0.7 °C higher than the long-term mean for these months. In contrast, precipitation was lower than the longterm value, rendering the growing season unfavourable for potato growth and development.
In 2013, the temperature was higher than the long-term mean and precipitation was by higher than the long-term value as much as 166.1 mm.
of samples taken earlier (expressed as t ha -1 ). The yield of large tubers consisted of tubers the diameter of which exceeded 50 cm and which were without any external or internal defects [Roztropowicz 1999]. Determination of the vitamin C content was carried out in fresh tubers 4-5 days after potato harvest. The vitamin C content was expressed in mg per 1 kg fresh matter. The vitamin C content was determined by means of Tilman's method as modified by Pijanowski [Rutkowska 1981].

Soil and meteorological conditions
The soil samples were taken each year before the experiment was set up from the topsoil at a depth of 0-30 cm. The soil was classified as Haplic Luvisol [IUSS Working Group WRB, 2015]. The soil was characterised by slightly acidic to

Statistical analysis
The data obtained in the experiment were analysed statistically using ANOVA. The significance of the sources of variation was tested with the F Fisher-Snedecor test, and the significance of differences between means was checked at the significance level p≤ 0.05 using Tukey's multiple intervals. All calculations were performed in MS Excel using the authors' own algorithms based on the split-plot mathematical model. The above-mentioned statistical procedures are presented in the work by Trętowski and Wójcik [1991]. The relationships between potato plant height, yield of large tubers, vitamin C content and the leaf greenness index SPAD were also determined by computing linear correlation coefficients.

Leaf greenness index SPAD
The SPAD leaf greenness index determined at the first date averaged 39.71 units and ranged from 34.87 to 43.70 (Tables 4, 5 In the study reported here, the leaf greenness index values were affected by the methods of herbicide and biostimulant application. These products increased the SPAD parameters compared with control, the highest readings resulting from a combined application of herbicides and biostimulants. A similar response was observed by Dvořák et al. [2016], whereas Wadas and Dziugieł [2020] found no influence of the following biostimulants: Bio-algeen S90, Means followed by the same letters do not differ significantly at P≤0.05. Means in columns marked with capital letters refer to interactions between the factors. Means in the last column and means in the last row (followed by lowercase) are for methods and cultivars.
Kelpak SL and HumiPlant on the SPAD values in potato leaves although they noticed a significant impact of study years on this characteristic. In the research discussed here, there was no influence of weather conditions nor interaction between years and methods of product application on SPAD determined at both dates (Table 5).

Potato plant height, yield of large tubers and the vitamin C content in potato tubers
Potato plant height, yield of large tubers and the vitamin C content were significantly affected by methods of herbicide and biostimulant application as well as cultivars, weather conditions having an effect on plant height only (Tables 6, 7). Means followed by the same letters do not differ significantly at P≤0.05. Means in columns marked with capital letters refer to interactions between the factors. Means in the last column and means in the last row (followed by lowercase) are for methods and cultivars. Means followed by the same letters do not differ significantly at P≤0.05. Means in columns marked with capital letters refer to interactions between the factors. Means in the last column and means in the last row (followed by lowercase) are for methods and years.
The tallest plants were recorded for cv. Gawin, cv. Bartek being the lowest. The highest large tuber yield and vitamin C content were found for cv. Honorata. They were lower for cv. Bartek and significantly lowest for cv. Gawin. The cultivarrelated differences for the discussed characteris- Herbicides applied alone or in combination with biostimulants contributed to an increase in plant height, yield of large tubers and the vitamin C concentration compared with control. The tallest potato plants and the highest vitamin C content in tubers were observed after a combined application of herbicides and biostimulants, the highest yield being harvested following spraying with Sencor and Sencor+Asahi SL. A positive effect of chemical control methods on tuber yield was reported by Barbaś  The weather conditions during the growing season significantly influenced plant height only ( Table 7). The tallest plants grew in 2012 and the lowest in 2013. It was also observed that, in terms of yield and vitamin C accumulation, precipitation and thermal conditions which were the closest to optimum prevailed in 2014, which is consistent with the findings of long-term research by Kalbarczyk and Kalbarczyk [2009]. Linear correlation coefficients confirmed that the leaf greenness index SPAD at both determination dates was strongly associated with plant height and large tuber yield of all the cultivars ( Table 8).
The vitamin C content was significantly correlated with SPAD I (the first date) for cv. Honorata, and SPAD II (the second date) for cv. Gawin and Honorata. Similar relationships were reported by other authors. Tang et al. [2018] found that SPAD was positively correlated with the Solanum tuberosum plant height under thermal stress conditions, Dvořák and Král [2019] observed a strong correlation of SPAD with tuber yield and weight Means followed by the same letters do not differ significantly at P≤0.05. Means in columns marked with capital letters refer to interactions between the factors. Means in the last column and means in the last row (followed by lowercase) are for metthods and years.

CONCLUSIONS
The findings of this study showed that herbicides and herbicides combined with biostimulants contributed to an increase in the leaf greenness index SPAD determined by a Konica Minolta SPAD-502Plus measuring device, plant height, yield of tubers and the vitamin C content in tubers. Strong correlation associations between SPAD and the determined characteristics (potato plant height, yield of large tubers and the vitamin C content) confirm a positive effect of the applied products and, simultaneously, they suggest SPAD may be a good indicator of normal plant development. Thus, the device measuring SPAD, as a non-invasive tool, may have a wider range of applications in integrated and precision agriculture.