The activity of β-glucosidase and guaiacol peroxidase in different genotypes of winter oilseed rape (Brassica napus L.) infected by Alternaria black spot fungi

The plants have developed several defense mechanisms to counteract pathogens. Among others, it includes activation of antioxidant enzymes like β-glucosidase and guaiacol peroxidase (GPX). These proteins participate in the oxidation of phenolic compounds, contributing to their increased fungitoxicity. The study aimed to analyze changes in the activity of β-glucosidase and GPX in four genotypes of winter oilseed rape (Mendel, Monolit, Polka, line L1425) inoculated with seven isolates: Alternaria brassicicola, Alternaria brassicae, Alternaria alternata (3 strains), Ulocladium chartarum (syn. A. chartarum), and Cladosporium cladosporioides. We noted that the varieties of oilseed rape, tested fungal species, and time of the plant material collection had significant (P < 0.001) effect on the activity of β-glucosidase and GPX per protein and fresh matter content comparing to the control group. A. brassicicola caused the highest mean increase in β-glucosidase and GPX activity in all examined genotypes, while other pathogens had a lower impact. Significantly lower β-glucosidase activity inoculated by various pathogens was noted between the L 1425 line and other varieties. GPX activity was in the opposite, the lowest activity was recorded in the Mendel variety, and the highest in the L 1425 line.


Introduction
Currently, a significant threat to rapeseed is Alternaria black spot. The infection significantly reduces the area of photosynthesis and accelerates senescence of the plant, and negatively affects the proper development and quality of seeds, its weight, color, and oil content (Kumar et al. 2014;Meena et al. 2010). The intensification of the disease occurrence is affected mostly by weather conditions, such as the amount and distribution of rainfall, temperature, and relative humidity of the air (Koch et al. 2007). In the conditions favorable for pathogens development, crop losses can reach up to several tens of percent (Koch et al. 2007). The intensity of Alternaria infection also depends on the rapeseed variety (El-Beltagi et al. 2011;Kumar et al. 2014). In Poland, the most common Alternaria black spot pathogens are A. brassicae, A. alternata, and A. brassicicola (Jajor and Korbas 2012). Many Alternaria species produce mycotoxins, important in the pathogenesis of plants. They can act by direct damage to plant tissue or by inducing programmed cell death (Logreico et al. 2009). The disease is often accompanied by other saprotrophic species re-inhabiting plant tissues like Ulocladium, Embellisia, and Cladosporium (Perek et al. 2017). However, their significance for the development of Alternaria black spot was not explained definitively.
Plants have developed different defense mechanisms to counter the attack of pathogens. The passive defense is based on the presence of structural barriers or anti-microbial factors preventing infection of plant tissues. In active protection, many enzymatic pathways are involved (Pandey et al. 2017). It includes activation of defense enzymes such as β-glucosidase or guaiacol peroxidase (GPX) (Prasannath 2017). These natural plant protection processes have an essential role in the crop plant defense to pathogens, in addition to varieties with genetically induced resistance.
In plants, β-glucosidases are involved in many physiological processes. Among others, they are responsible for the formation of intermediates in cell wall lignification, cell wall degradation, activation of phytohormones, biotic and abiotic stress response or stimulation of defense compounds (Ashraf and Ali 2008;Halkier and Gershenzon 2006;Ketudat Cairns and Esen 2010;Lee et al. 2006). In the infection response, β-glucosidases are involved in the release of glucose from oligosaccharides, hydrolysis of glucosinolates, cell wall catabolism, activation of lignin precursors, the release of phytohormones or toxic compounds, such as aldehydes or ketones (Ketudat Cairns and Esen 2010;Moller 2010;Morant et al. 2008;Rask et al. 2000;Rojas et al. 2018;Sherameti et al. 2008).
The plant peroxidases are associated with many biosynthetic processes of plant growth, including cell wall metabolism, lignification, fruit growth and ripening, seed germination, and defense against abiotic and biotic stresses. (Passardi et al. 2005). These stress factors include different pollutants, salt, herbicides, ozone, or pathogen pressure (Heidari 2010;Sharma et al. 2012). GPX is involved in removing excess reactive oxygen species such as hydrogen peroxide (H 2 O 2 ) and can lower the free radical generation and neutralize excess free radicals created by stress conditions (El-Beltagi et al. 2011). Moreover, it participates in the oxidation of phenolic compounds, contributing to their increased fungitoxicity. Thus it generates metabolites for the synthesis of structural barriers: lignins, melanins, feruloylated polysaccharides, and glycoproteins, etc. limiting the spread of pathogenic microorganisms in plants (Beckman 2000;Vance and Kirk 1980).
To date, the influence of infection with fungal pathogens on the activities of β-glucosidase or GPX occurring in various rapeseed varieties has not been considered enough. The study aimed to analyze changes in the activity of β-glucosidase and GPX in different varieties of winter oilseed rape inoculated with pathogens causing Alternaria black spot-A. brassicicola, A. brassicae, A. alternata, as well as Ulocladium chararum, and Cladosporium cladosporioides.

Plant material
For the study, four winter oilseed rape varieties: Monolit, Mendel, Polka, and a yellow-seeded line L 1425 were used. Monolit is the most common open-pollinated cultivar in Poland. It is characterized as cold-resistant, high seed yield, and high oil content. Mendel is the first Polish hybrid variety with confirmed resistance to basic types of clubroot. Polka is a new open-pollinated variety included in the Polish National List of Varieties in 2016, characterized by a modified fatty acid composition ratio (over 70% of oleic acid and a reduced content of linoleic acid). Line 1425 is a yellow-seeded line, with higher oil and protein content and lower fiber content in seeds in comparison to black-seeded genotypes.

Fungal isolates
In the study, Alternaria brassicicola, Alternaria brassicae, Alternaria alternata (three genetically different isolates), Ulocladium chartarum (syn. A. chartarum), and Cladosporium cladosporioides were tested. The fungal cultures were isolated in 2016 from infected oilseed rape plants derived from fields located in the region of Greater Poland (Wielkopolska). Species of tested isolates were identified based on macro-and microscopic morphological features of the colonies (Simmons 2007). Morphological identification was confirmed by sequence analysis of ribosomal DNA (ITS1-5.8SrDNA-ITS2).
All tested isolates were spiked on PDA (potato dextrose agar) or PCA (potato carrot agar) medium in Petri dishes. The colonies were incubated at room temperature (20-24 °C) for 2 weeks. Then spores were collected from the surface of the medium and made into inoculum slurry.

Greenhouse experiment
Each genotype of rapeseed was sown in 72 pots (280 cm 3 ) filled with Kronen gardening soil (pH 6.0-6.8). Ten seeds were sown in each pot to obtain five plants per pot as a result. The containers were placed in the greenhouse at 26-28/14-16 °C (day/night) with natural light and watered by the accepted practice for the species. After 32 days, the plants were inoculated by spraying the upper surface of leaves by different pathogen spores: A. brassicicola (A), A. brasicae (B), three genetically different isolates of A. alternata-C, E, G (based on ITS1-5.8SrDNA-ITS2 sequence analysis), U. chartarum (D), C. cladosporioides (F). The spore concentration was determined with the Thoma cell counting chamber and standardized to 0.5 × 10 6 /ml, except for C. cladosporioides which concentration was 2 × 10 6 /ml. Each inoculum in the amount of 50 ml was applied equally to 36 pots (four genotypes × three date of sample collection × three replications of experiment). The last set of 36 pots was sprayed with clean water and treated as a control group (K). The pots with inoculated plants were covered with transparent plastic bags to maintain stable and high air humidity (> 85%). For the study of enzymes activity, leaf samples (about 0.2 g) were collected on the third (T1), sixth (T2), and ninth (T3) day after inoculation, in three replications. The samples were placed in Eppendorf tubes (2 ml) and deep-frozen (− 80 °C) until analysis.

Enzymatic assays
The activity of β-glucosidase was determined based on Nichols et al. (1980). The leaves were ground in 0.1 M phosphate buffer pH 7.0 containing 0.5% of polyethylene glycol and 40 mg of Polyclar AT. The supernatant obtained after centrifugation at 10,000g for 15 min at 4 °C was used to determine enzyme activity. The mixture containing 0.3 ml of extract and 0.3 ml of 4-nitrophenyl-b-d-glucopyranoside as a substrate was incubated for 1 h at 30 °C. After the time, 0.9 ml of 0.2 M Na 2 CO 3 was added. The formation of p-nitrophenol (p-NP) was followed at 400 nm. The activity was expressed as μmoles p-NP per mg protein (ActGluProt) and per mg of fresh matter (ActGluFM).
Guaiacol peroxidase measurement was based on the method of Hammerschmidt et al. (1982). Leaves were homogenized in 0.1 M K-phosphate buffer pH 7.5 and centrifuged at 10,000g for 15 min at 4 °C. Enzyme assays were prepared by adding 0.5 ml 0.1 M K-phosphate buffer pH 7.5, 0.5 ml extract, 0.5 ml 3.4 mM guaiacol, and 0.5 ml H 2 O 2 to a glass cuvette. The absorbance at 480 nm was measured, and the guaiacol oxidation was expressed as nanokatals per mg protein (ActGPXProt) and per mg of fresh matter (ActGPXFM).
The determination of protein content in the leaves was made with the help of Bradford's (1976) and Kruger (2009) method. 2 ml of a solution of Coomassie Brilliant Blue G-250 (CBB) in 85% orthophosphoric acid was added to 100 μl of a diluted extract. After 10 min the absorbance was measured at a wavelength of 595 nm. Protein content was determined from a curve plotted for albumin.

Statistical analysis
The normality of the trait's distribution was tested using the Shapiro-Wilk's normality test (Shapiro and Wilk 1965). The three-way fixed model analysis of variance (ANOVA) was carried out to determine the effects of varieties of oilseed rape, pathogens, and term of sampling, as well as all interactions on the variability of observed traits (ActGluProt, ActGluFM, ActGPXProt, ActGPXFM). Estimations were also made by mean values, maximum and minimum values, and the coefficient of variation for the studied traits (Kozak et al. 2013). When critical differences were noted, multiple comparisons were carried out using the least significant differences (LSDs) for each trait. Based on this, homogeneous groups (not significantly different from one another) were determined for the analyzed traits. The relationships between observed traits were estimated using Pearson's correlation coefficients (Kozak et al. 2010) and presented in the scatterplot matrix. Data analysis was performed using the statistical package GenStat 17.

Disease symptoms
In the conducted experiment at T1, all cultivars treated with A. brassicicola (A) on leaves exhibited first symptoms of infection-small, single dark spots. At T2, the spots were more numerous and also appeared on stalks; at T3, the disease symptoms were observed on about 80% of the plants. In all tested cultivars inoculated with A. alternata (E and G) and also C. cladosporioides (F) and U. chartarum (D), the first disease symptoms appeared in T2. In T3, about 20% of plants presented symptoms of infection by A. alternata (E and G). In groups treated with A. brassicae (B) and A. alternata (C) in T3, only scarcely single plants were infected.

β-glucosidase activity/pathogens
The highest mean β-glucosidase activity in samples collected in T1 was observed in plants inoculated with A. brassicicola (A) (mean 1.23 μmoles p-NP/protein) (  Fig. 1). The enzyme values measured in samples inoculated with other pathogens ranged from 1.40 to 1.11 μmoles of p-NP/mg protein. The lowest enzyme activity value (0.75 μmoles p-NP/mg protein) was reported in the control samples in all terms.

GPX activity/pathogens
The highest average GPX activity in T1 was observed in plants inoculated with A. brassicicola (A) (2.42 nanokatals/ mg protein) (  Fig. 2). The enzyme values determined in the rest of the samples ranged from 3.88 (A. alternata G) to 2.65 (U. chartarum D) nanokatals/mg protein.

GPX activity/rapeseed varieties
The highest GPX activity in T1 was observed in the Monolit variety after inoculation with A. brassicicola (A)-3.30 nanokatals/mg protein in the first term of collecting samples (Table 2; Fig. 2). In the Polka variety, the highest activity of GPX was measured after inoculation with C. cladosporioides (F)-2.80 and in the line 1425 after   (Table 2; Fig. 2). Inoculation of tested varieties with other pathogens caused variations in enzyme activity. In T2, the highest activity of GPX was observed in the Monolit and Polka samples inoculated with A. brassicicola (A) (Table 2; Fig. 2). These values amounted to 3.35 and 3.13 nanokatals/mg protein, respectively. In L 1425 plants, the highest activity of the enzyme was induced by inoculation with A. alternata (G)-3.77 nanokatals/mg protein. In T3, the impact of different species of fungi on GPX activity was varied. The highest GPX activity was recorded for the Mendel cultivar (6.61) and L 1425 line (6.37 nanokatals/mg protein) after inoculation with A. brassicicola (A) (Table 2; Fig. 2). A high level of enzyme activity was observed for Polka as a result of inoculation with A. brassicicola (A)-4.25, and for a yellow-seeded line with A. brassicae (B)-4.64 nanokatals/mg protein.
The three-way analysis of variance also demonstrated the significance of the interaction: variety/pathogen, variety/day after infection (DAI), pathogen/DAI, and variety/ pathogen/DAI (Table 3). The activity of β-glucosidase and GPX per the protein content and fresh matter were positively correlated at P < 0.001 (Table 4).

Discussion
We noted that the varieties of oilseed rape, tested fungal species, and time of the plant material collection had significant (P < 0.001) effect on the activity of β-glucosidase and GPX per protein and fresh matter content comparing to the control group. However, the indication of one factor with the highest effect on the increase in the activity of the enzymes is difficult.
One of the most interesting factors affecting the increase in enzyme activity was the species of fungus used for inoculation. The highest average increase in β-glucosidase activity in all tested cultivars was caused by the most pathogenic species A. brassicicola. In the Mendel and L 1425 cultivars, it was particularly visible in T3 (2.02 and 1.97 μmoles p-NP/mg protein, respectively) while in the Monolit cultivar in T1 and T2 (1.47 and 1.48 μmoles p-NP/mg protein,  . These results may indicate significant differences in β-gluosidase activity or time of enzymatic response for the pathogen infection between tested cultivars. Significant variations in phytochemical constituents among different rapeseed cultivars were previously reported by El-Beltagi et al. (2011). The highest GPX activity in all tested cultivars was also observed for A. brassicicola inoculation mainly in T3 (mean 5.38, 4.64 nanokatals/mg protein), but the activity increased gradually. Presumably, the GPX results were associated with the full development of the disease symptoms. Interesting observations relate to enzymatic changes occurring under the influence of A. alternata inoculation. A. alternata is the most cosmopolitan fungal species and causes diseases in over a hundred varieties of host plants. The species contains strains of varied pathogenicity and saprotrophs (Coates and Johnson 1997;Woudenberg et al. 2015). It can explain the obtained differences in enzyme activity. In the study, three genetically distinct A. alternata isolates were used. By analyzing average activity values, the isolate C of this pathogen induced a slightly weaker increase in β-glucosidase (except Mendel) (T1-1.41, T2-2.19, T3-2.87 μmoles p-NP/mg protein) and GPX activity (T1-1.41, T2-2.19, T3-2.87) than E (β-glucosidase: T1-1.41, T2-2.19,T3-2.87 μmoles p-NP/mg protein; GPX: T1-1.41, T2-2.19, T3-2.87 nanokatals/ mg protein) and G (β-glucosidase: T1-1.41, T2-2.19, T3-2.87 μmoles p-NP/mg protein; GPX: T1-1.41, T2-2.19, T3-2.87 nanokatals/mg protein) isolates. Isolate C was less pathogenic then E and G. In all tested cultivars inoculated with A. alternata (E and G) first disease symptoms appeared in T2 while in T3 isolate C infected only single plants. So, except for a few cases, the results correlate with enzyme activity. Variability of responses within one potato cultivar to different isolates of A. solani was described by Shahbazi et al. (2010). The authors indicate that the presence of genotypic diversity between isolates results in the diversity of the pathogen aggressiveness. Another main oilseed rape pathogen A. brassicae (B) is very changeable in induced enzyme activity. Attention is drawn only to a high level of β-glucosidase and GPX activity in Mendel variety in T3 (2.02 μmoles p-NP/mg protein and 5.93 nanokatals/ mg protein) and GPX in Polka in T3-3.57 nanokatals/ mg protein. Low pathogenicity of the strain A. brassicae can be explained by a successful response of plants to the pathogen, not enough time for the infection or influence of other factors. Typically, low β-glucosidase and GPX activity were observed by inoculation with less pathogenic species U. chartarum (D). Ambiguous results were obtained after inoculation with C. cladosporioides isolate (F). In some combinations, mainly in T1, the species caused a significant increase in β-glucosidase activity (Mendel T1, Polka T1, and T2). Additionally, C. cladosporioides isolate caused high GPX activity in the Polka variety in all three observation Table 2 Mean values of activity of GPX in nanokatals per mg protein (ActGPXProt) in three terms of collecting samples (T1, T2, T3) Values determined identical letters in columns not differ significantly for least significant differences (LSD  phases. This species is considered rather a saprotrophic, but in our study, it also inducted unexpected disease symptoms. There is not enough information describing changes in the level of β-glucosidase and GPX activity in crop plants under the influence of pathogens. Significantly higher peroxidase activity in potato treated by A. solani isolates was correlated with resistant cultivar (Shahbazi et al. 2010). Rojas et al. (2018) suggest the transcriptional regulation of β-glucosidases and their possible impact in the defense mechanism against fungal Fusarium proliferatum infections in maize. Plants also respond to bacteria (Ralstonia  Table 3 Mean squares form analysis of variance (ANOVA) of observed activity of β-glucosidase and guaiacol peroxidase and their interaction for a variety of rapeseed, pathogen and the term of collecting samples ActGluProt the activity of β-glucosidase in μmoles p-NP per mg protein, ActGluFM the activity of β-glucosidase in μmoles p-NP per mg of fresh matter, ActGPXProt the activity of guaiacol peroxidase in nanokatals per mg protein, ActGPXFM the activity of guaiacol peroxidase in nanokatals per mg of fresh matter, ns not significant ***P < 0.00  (Prakasha and Sharan 2016). The differences in the activity of both enzymes were visible between the tested varieties. Significantly lower β-glucosidase activity due to inoculation with various pathogens was noted between the L 1425 line and others, especially Mendel. However, GPX activity was the opposite, the lowest activity was recorded in the Mendel variety, and the highest in the L 1425 line. The results obtained may indicate that the tested varieties have different genetic characteristics that affect the response time to pathogen infection. The increase in GPX in plant tissues due to pathogen infection may be correlated with the level of plant resistance. Doullah et al. (2006) revealed different responses of different Brassica rapa genotypes to A. brassicicola infection, also under several factors-inoculum concentration, leaf stage, and incubation temperature. Differences in Fusarium head blight (FHB) resistance connected with enzymatic activity, among wheat cultivars were described by Spanic et al. (2020). The authors suggested that GPX activity, which is induced in response to pathogen invasion, might help the resistant wheat cultivar to limit infection by Fusarium species. Another study describes the response of different tomato genotypes to early blight disease caused by A. solani. The activation of defense responses-activities of catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase was also more visible in resistant and moderately resistant tomato genotypes (Nafisa Shoaib et al. 2020). Several factors determine a different level of disease resistance in rapeseed. Firstly, a presence of resistance genes and, secondly, the physical parameters of the plants which hinder pathogen access and penetration of plant tissues (El-Beltagi et al. 2011). The effectiveness of enzymatic pathways like β-glucosidase and GPX can contribute to maintaining plant health. The significant differences in β-glucosidase and GPX activity between tested varieties of rape in response to inoculation with different species of pathogenic fungi may indicate the diversity of the variety's capabilities to withstand the pathogen infection. The significant differences in β-glucosidase and GPX activity between tested varieties of rape in response to inoculation with various species of pathogenic fungi may indicate the unequal capabilities of particular varieties to withstand the pathogen infection. The study also showed significant differences in the activity of the enzymes for the inoculation of strong and weak pathogenic fungi.
Author contribution statement KP designed the research and analyzed the experimental results. KP, AD, JW and AP carried out experiments in the greenhouse. MZ and MRZ carried out enzymatic tests. JB conducted a statistical analysis and assisted in analyzing the data and interpreting the results. KP, AD, JW, MRZ and JB wrote the article. KP and MRZ corrected the manuscript.
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