Introduction

In Egypt, sugar beet (Beta vulgaris L.), which is ranked as the second-largest sugar crop after sugar cane, is a crop of the temperate regions (Eweis et al., 2006 and Amer et al., 2019).

Many pathogenic fungi affect sugar beet plants leading to severe disease and yield loss. The most devastating foliar disease of sugar beet, Cercospora leaf spot (CLS) is caused by Cercospora beticola Sacc., damages the leaves, negatively affects plant photosynthetic ability, induces biochemical changes in amino acids, phenols, and sugar that decrease sugar yields to 30 and 50%, respectively (Wolf & Verret, 2002; Wolf & Vereet, 2005; Weiland & Koch, 2004; Kaiser et al., 2010 and Skaracis et al., 2010).

CLS reduces root and extractable sucrose yields and raise impurity concentrations, which increases processing losses (Farahat, 2018 and Morsy et al., 2022).

The main methods of disease control include the use of fungicides, the development of resistant cultivars, and crop rotation (Morsy et al., 2022; Tedford et al., 2019). Synthetic fungicides have been applied repeatedly and widely over the past ten years with detrimental effects to the environment and human health.

Biological control as an ecofriendly method providing a logical substitute for synthetic fungicides for the control of various diseases (Bharathi et al., 2004; Galletti et al., 2008 Shahraki et al., 2008; Jacobsen, 2010 and Derbalah et al., 2013).

Many bacterial isolates of Bacillus spp. and fungal isolates of Trichoderma spp. produce antibiotics enzymes, and show mycoparasitism, where a strain of fungus or bacterium preys on other fungi (Harman, 2000). Due to the elicitation of systemic resistance, repeated applications of Bacillus spp. decrease sugar beet CLS symptoms under field conditions (Bargabus et al., 2002).

Different bioagents, including Trichoderma harzianum, T. viride, Gliocladium virens, Bacillus subtilis, and Pseudomonas fluorescens, are successful in reducing disease incidence and increase yield when compared to control treatments (Srivastava, 2004; Patel & Jasrai, 2012; Ray & Swain, 2013 and Sharma, 2015). Trichoderma spp. produce lytic enzymes such as chitinases, peroxidases, polyphenoloxidases, and glucan 1–3 -glucosidases that damaged the pathogen's cell wall. B. subtilis creates a collection of enzymes that decrease the strength of the pathogen's cell wall, and produce volatile chemicals, phytotoxic substances, and antibiotics like bacterocin and subtilisin (Jacobsen et al., 2004 and Muthuvelayudham & Viruthagiri, 2006).

In vitro, Trichoderma koningii (T1), Bacillus subtilis (B1), and Bacillus subtilis (B2) have a strong antagonistic effect on Cercospora beticola. In addition, increased enzyme activities of peroxidase (POX), catalase (CAT), and polyphenol oxidase (PPO) reduce the severity of CLS. As a result of using the product Eminent's superior control, root productivity, total soluble solids (T.S.S.), sucrose percentage, and chlorophyll content improved (Chen et al., 2022 and Hamden et al., 2023).

The purpose of this study was to evaluate the effectiveness of specific recommended biocontrol agents to control Cercospora leaf spot on sugar beet under organic field conditions and to identify which application regimen could be used to manage the disease, produce sugar with high quality and quantity without toxicity at the food chain.

Material and methods

Plant components

Seeds of Sugar beet cv. Gloria were obtained from the Sugar Crops Research Institute (SCRI), Agricultural Research Center, ARC, Giza Governorate, Egypt. Field tests were conducted throughout 2020/2021 and 2021/2022 at Fayoum Governorate, Egypt.

Biological antagonists

Trichoderma harzianum, T. album, Bacillus subtilis and B. megaterium were added at the rate of 1 Lit./50 Lit. water. These biocontrol organisms were kindly provided by the Biological Control Production Unit, Central Laboratory of Organic Agriculture, CLOA, ARC.

Biocide products

The biocide formulations were used as a comparison with other treatments as follow:-

  • Blight Stop is recommended as biocide preparation. It contains Trichoderma harzianum (30 × 106 spore/ml). This product was used at a rate of 1 Lit./50 Lit. water and was kindly provided by the Biological Control Production Unit Central Lab. of Organic Agriculture, CLOA; ARC.

  • Root Guard, a biocide preparation that was kindly provided by the Biological Control Production Unit Central Lab of Organic Agriculture, CLOA; ARC is recommended. It contains Bacillus subtilis (30 × 106 cell/ml) and is used at a rate of 1 Litre per 100 Litres of water.

  • Bio Zeid 25% WP, which was provided by Kafr El Zayat Pesticides and Chemicals (kz), is recommended as a biocide preparation. It contains T. album (2.5% w/w) and is used at a rate of 250 g/100 L water.

  • Bio ARC 6% WP, a biocide preparation that was kindly provided from kz firm is a recommended control product. It contains B. megaterium (6% w/w) and is used at a rate of 250 g/100 L water.

Isolation of Cercospora leaf spot

In a private organic farm in the Fayoum Governorate, during the 2020–2021 season, Cercospora leaf spot disease was discovered in sugar beet cv. Gloria leaves. 20 infected leaves were collected, washed carefully with tap water, cut into small pieces, surface sterilized in 3% sodium hypochlorite solution for three minutes, repeatedly washed in sterile distilled water, dried between two sheets of sterile filter paper (v/v) and transferred onto Petri dishes (9 cm) containing sugar beet leaf extracts dextrose agar medium (SBLEDA). The fresh sugar beet leaves sliced and 200 g was boiled in one liter distilled water for 15 min and strained through double layers of cheese cloth. The SBLEDA medium consists of sugar beet leaf extract (100 ml), dextrose (20 g), and agar (15 g) medium. Plates were incubated at 27 ± 2 °C for 3–7 days and examined daily for occurrence of fungal growth. Fungal growth was examined microscopically and then purified using the hyphal tip method (Dhingra & Sinclair, 1995 and Morsy et al., 2022). Pure cultures of each isolate were maintained on PDA at 4 °C for further examination.

Identification of Cercospora leaf spot

Identification according to the fungal cultural, phytopathological, and microscopic traits (Alexopoulos et al., 1996). Based on identification, one of them was chosen for further examination.

Antagonistic effect of bacterial strains against Cercospora beticola

The antagonists and the commercial preparation suspensions were added to warm sterilized PDA medium at the rate of 10% and poured before solidification into Petri dishes (10 ml/plate). After solidification, a disc (5 mmØ) of Cercospora beticola obtained from the periphery of 7-days old mycelium on the same medium was placed in the center of each plate. Plates containing media without antagonists and inoculated only with abovementioned pathogen served as control treatment. Three plates were used for each treatment. Inoculated plates were incubated at 27 ± 2 °C. The experiment was terminated when mycelial mats covered the surface in the control treatment, all plates were examined and the percentage reduction in mycelial growth of the fungus was calculated using the formula suggested by Ahmed (2005) and Ahmed (2013) as following:

$$\mathbf{\%}\mathbf{R}\mathbf{e}\mathbf{d}\mathbf{u}\mathbf{c}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n}\,\mathbf{i}\mathbf{n}\,\mathbf{l}\mathbf{i}\mathbf{n}\mathbf{e}\mathbf{a}\mathbf{r}\,\mathbf{g}\mathbf{r}\mathbf{o}\mathbf{w}\mathbf{t}\mathbf{h}\,\mathbf{o}\mathbf{f}\,\mathbf{p}\mathbf{a}\mathbf{t}\mathbf{h}\mathbf{o}\mathbf{g}\mathbf{e}\mathbf{n}\mathbf{i}\mathbf{c}\,\mathbf{f}\mathbf{u}\mathbf{n}\mathbf{g}\mathbf{i}=\frac{\mathrm{G}1-\mathrm{G}2}{\mathrm{G}1}\times 100$$

where: G1: growth of the pathogenic fungus in control only, G2: growth of the pathogen against the tested antagonists.

Field trials

In Vivo studies were carried out at a private organic farm, Fayoum Governorate, Egypt, which has a long history of severe infection by Cercospora leaf spot disease (CLS) The experiments assess the effectiveness of different antagonists for the control of Cercospora beticola. Three replicated plots were used in a complete randomized block design for all of the trials. The experimental plot's surface area was 21 m2, made up of 3 rows (6 m × 50 cm) 50 cm apart. Each row was sown with 30 Sugar beet seeds cv. Gloria and were exposed to natural inoculum only. Sugar beet seeds cv. Gloria were planted on 15th October during the two growing seasons, 2020/21 and 2021/22, respectively.

The cultivation, irrigation and compost fertilization, were applied in an equal amount to every plot 15 days before sowing. After 90 days of cultivation, sugar beet plants were sprayed with suspensions of the four bioagent isolates—Trichoderma harzianum, T. album, Bacillus subtilis, and B. megaterium, as well as with the four commercial biocide products, including Blight Stop, Bio Zeid, Root Guard, and Bio ARC at the recommended doses as mentioned above. Super film as a surfactant and sticker material, was mixed with each treatment prior to spraying at a rate of 50 ml/100 L water. All treatments were applied twice with 15 days intervals between each application. Plots that had not been treated (only water sprayed) served as the control.

The following characteristics were evaluated:

Disease severity (DS %) and disease incidence (DI):

14 days after the last treatment disease was scored using the scale developed by Verreet et al. (1996), where 0 = no symptoms; 1 = 1–20%; 2 = 21–40%; 3 = 41–60%; 4 = 61–80%; and 5 = 81–100% infected leaf area, The following formula was used to calculate each foliar disease's severity percentage:

$$\mathbf{D}.\mathbf{S}.\mathbf{I}\mathrm{\%}=\frac{\sum (\mathrm{n}\times \mathrm{c})}{\mathrm{N}\times \mathrm{C}}\times 100$$

where, D.S.I = disease severity index, n = number of leaves in each category, c = numerical value of each category, C = numerical value of highest category and N = total number of leaves in the sample.

The percentages disease incidence and the efficacy of the tested treatments were estimated using the following equations:

$$\mathbf{D}\mathbf{i}\mathbf{s}\mathbf{e}\mathbf{a}\mathbf{s}\mathbf{e}\mathbf{I}\mathbf{n}\mathbf{c}\mathbf{i}\mathbf{d}\mathbf{e}\mathbf{n}\mathbf{c}\mathbf{e}(\mathbf{D}\mathbf{I})\mathbf{\%}=\frac{\mathrm{No}.\mathrm{ of diseased plants}}{\mathrm{Total No}.\mathrm{ of examined plants}}\times 100$$
$$\mathbf{E}\mathbf{f}\mathbf{f}\mathbf{i}\mathbf{c}\mathbf{a}\mathbf{c}\mathbf{y}\mathbf{\%}=\frac{\mathrm{ Control}-\mathrm{Treatment}}{\mathrm{ Control}}\times 100$$

Quality control criteria:-

  • 1-Sugar beet total yield

The average root weight per fed. area was evaluated (Mahmoud et al., 2012).

  • 2-Sugar, root, and top yield of sugar beet plants (ton/fed.)

The yield tops and roots (ton/fed.) was calculated at harvest. Root yield was multiplied by sucrose % to calculate sugar yield (ton/fed.). According to the method described by McGinnus (1982), quality parameters such as sucrose percentage and impurity content (K, Na, and alpha-amino N %) were measured by the Faiyum Sugar Works Company, Fayoum Governorate. Juice purity was determined using the formula provided by Devillers (1988).

$$\mathbf{P}\mathbf{u}\mathbf{r}\mathbf{i}\mathbf{t}\mathbf{y}\mathbf{\%}=99.36-\left(14.27\times \left(\frac{ \sum (\mathrm{Na }+\mathrm{ K}+\mathrm{ \alpha }-\mathrm{N})}{\mathrm{Sucrose\%}}\right)\right)$$

  • 3-Total soluble solids (T.S.S %)

Total soluble solids (T.S.S %) in fresh roots were evaluated using a manual refractometer according to McGinnis (1982).

  • 4- Chlorophyll content

The total chlorophyll content was determined using method of Moran (1982).

Biochemical changes in sugar beet plants due to application of chemicals

Total phenol

The amount of phenol was calculated using the typical graph made with different catechol concentrations. The catechol equivalents of the phenol content were calculated as mg/g of fresh tissue (Turkmen et al., 2005).

Statistical analysis

All data collected over two successive seasons were statistically analysed and compared using the least significant difference (L.S.D.) at 5% proposed by Snedecor and Cochran (1989).

Results

Effect of different antagonists on the growth of Cercospora beticola

The data in (Table 1) demonstrate that the antagonists’ inhibited the growth of Cercospora beticola in vitro. T. harzianum greatly reduced mycelial growth by 89.60%, followed by T. album (87.50%). The smallest control was found with Bio ARC (68.10%)

Table 1 Effect of the antagonists on the percentage reduction in growth of Cercospora beticola after incubation at 27 ± 2 °C for 5 days
Full size table

The effect of different antagonists on sugar beet Cercospora leaf spot under field conditions during the 2020/21 and 2021/22 growing seasons:

  • 1. Diseases parameters

The results in (Table2) indicate that, all tested biological control treatments (Trichoderma harzianum, T. album, Bacillus subtilis, B. megaterium, Blight Stop, Bio Zeid, Root Guard, and Bio ARC) significantly outperformed the control treatment in reducing the incidence and severity of sugar beet Cercospora leaf spot disease in the two growing seasons. Blight Stop biocide showed the best efficacy (78.72 and 89.43%), followed by T. harzianum isolate (77.33 and 86.37%). On the other hand, B. megaterium had the lowest effectiveness (68.62 and 68.98%) in controlling the Cercospora beticola leaf spot disease in both seasons compared with control treatment.

Table 2 The effect of different antagonists on disease incidence and severity of sugar beet Cercospora leaf spot under field conditions during 2020/21 and 2021/22 growing seasons
Full size table

  • 2. Total phenols, total chlorophyll and total amino acids content

Presented data in (Table 3) illustrate that all t biological treatments increased total chlorophyll and phenols in comparison with untreated plants during both growing seasons. The biocide Blight Stop provided the highest level of total chlorophyll (79.70 and 80.11) and phenols (10.19 and 10.25) in the majority of cases, followed by T. harzianum. In contrast, B. megaterium demonstrated the smallest effectiveness (72.27 and 73.17 in total chlorophyll and 6.30 and 6.32 in total phenols).

Table 3 The effect of different antagonists on total chlorophyll, phenols and amino acids under field conditions during the 2020/21 and 2021/22 growing seasons
Full size table

Compared to the control treatment, all biological treatments reduced the total amino acid consentration in sugar beet roots throughout the two growing seasons. Untreated plants had the highest levels of amino acids in both growing seasons. Amino acid levels were found to be lowest in sugar beet plants that had been treated with Blight Stop. B. megaterium produced the highest level of amino acid content.

  • 3. Quality of commercial sugar beet production and juice impurities (K, Na and α-amino N %):

The results, which are presented in (Table 4) show that, as compared to the untreated control, all tested treatments considerably reduced juice impurities (K, Na and -amino N%) in comparison with the control treatment. The Blight Stop treatment had the highest reduction in juice impurities followed by Trichoderma harzianum during the two successive growing seasons compared with control treatment.The B. megaterium treatment was the least effective in reducing impurities.

Table 4 The effect of different antagonists on juice impurities (Na, K and α-amino N %) under field conditions during the 2020/21 and 2021/22 growing seasons
Full size table

  • 4. Total soluble solids (TSS), Sucrose % and Juice purities:

The data (Table 5) demonstrate that all tested biological treatments significantly increased total soluble solids (TSS), sucrose, and purity percentage when compared to the untreated plants. Spraying Blight Stop resulted in the largest increase in TSS, sucrose, and purity during both seasons, followed by T. harzianum. In contrast, B. megaterium was the least effective treatment compared to the untreated control during the two growing seasons 2020/21 and 2021/22.

Table 5 The effect of different antagonists on TSS, sucrose and juice purities under field conditions during 2020/21 and 2021/22 growing seasons
Full size table

  • 5. Root, top and sugar yields:

Regarding the influence of biological treatments on root and recoverable sugar yields/fed. (Table 6), all tested treatments significantly increased root, top and sugar yields throughout both seasons compared with untreated plants.

Table 6 The effect of different antagonists on root, top and sugar yields of sugar beet plants under field conditions during 2020/21 and 2021/22 growing seasons
Full size table

The most superior treatments increased the root, top and sugar yields was observed when sugar beet plants were sprayed with Blight Stop twice, followed by T. harzianum compared to the other treatments. The B. megaterium treatment showed the least effect on in root, top and sugar yields in comparison with the control treatment.

Results in Tables 4, 5 and 6 shows that, in comparison to the untreated control all tested treatments resulted in high values for the quality traits (sucrose and purity percentages), root, top and sugar yields of sugar beet, and lowest juice impurity. This discovery may be attributed to a decline in impurities, such as sodium, potassium, and alpha amino-N levels, as well as a decline in disease incidence and severity, which had an impact on root production and sugar content.

Discussion

In recent years, farmers have become aware that using chemical pesticides may be harmful to the environment and to human health and may contribut to the emergence of new pests, reduce the number of natural enemies. In order to produce high-quality food in sufficient quantities and enhance biodiversity, the current work aimed to reduce the use of toxic chemicals in agriculture. In addition, we aimed to find the best non-chemical methods. to protect sugar beet plants from Cercospora beticola leaf spot disease.

Data in Table 1 showe that, T. harzianum greatly reduced mycelial growth, followed by T. album, Blight Stop "T. harzianum", Bio Zeid "T. album", B. subtilis, Root Guard "B. subtilis", and B. megaterium. Bio ARC's "B. megaterium" had the least impact.. Bacillus subtilis held the second position after Trichoderma spp., this may be because it generates a group of enzymes that degrate the pathogen's cell wall (Ahmed, 2005 and Ahmed, 2013), antibiotics such as bacterocin and subtilisin (Hamden et al., 2023), volatile compounds and phytotoxic substances (Stein, 2005). This phenomenon could be explained by the fact that different infections with distinct striations have unique defence mechanisms against the enzymes and toxic materials produced by different antagonists. (Ahmed, 2005, 2013; Muthuvelayudham & Viruthagiri, 2006). Trichoderma species produce lytic enzymes such as chitinases, peroxidases, polyphenoloxidases, and glucan 1–3 B-glucosidases that destroyed the pathogen's cell wall (Jacobsen et al., 2004 and Derbalah et al., 2013). Furthermore, Trichoderma are known to be able to cause systemic acquired resistance (SAR), and this is thought to be one of the most crucial modes of action for this biocontrol agent. This has been described for a range of plant-pathogen systems (Harman et al., 2004).

According to the available data (Table 2), all tested biological control treatments (Trichoderma harzianum, T. album, Bacillus subtilis, B. megaterium, Blight Stop, Bio Zeid, Root Guard, and Bio ARC) significantly outperformed the control treatment in reducing the incidence and severity of sugar beet cercospora leaf spot disease in the two growing seasons of 2020–2021 and 202–2022. The results can be evaluated using both the chemical impact of antioxidants, which clearly improve plant physiology and metabolism and induce systemic resistance (ISR), and the action of biotic factors, which provide growth regulators (Harman et. al., 2004). Due to competition for oxygen, nutrients, and space as well as their capacity for fungal mycoparasitism (El-Sayed et al., 2017) and the secretion of antibiotics, Trichoderma spp. and/or Bacillus spp. have direct effects on pathogenic fungi (Patel & Jasrai, 2012, and Sharma, 2015). Trichoderma spp. can also create antifungal compounds such as trichodermin, alpha-1,3-glucanase, beta-glucosidase, and endo chitinase (Galletti et al., 2008, Stefania et al., 2008 and Hamden et al., 2023).

The biocide Blight Stop provided the highest levels of total chlorophyll and phenols in the majority of cases, followed by T. harzianum as shown in Table 3. In order to reduce the pathogen's harmful effects, bioagents may provide the nutrients and biological elements needed to enhance photosynthesis in the host plants which decrease disease incidence and severity (%), decrease the loss of photosynthetic leaf area, and decrease toxicity from Cercosporin-related toxins, which have a negative impact on plant health and photosynthesis (Scholes & Rolfe, 2009 and El–Mansoub et al., 2017). Cercospora beticola infection of sugar beet impacts chlorophyll and phenol concentration even before causing symptoms, and imaging raw chlorophyll fluorescence allowed for presymptomatic detection of the necrotrophic fungal disease, C. beticola, in sugar beet leaves (Wolf & Vereet, 2005 and Chaerle et al., 2007). The biodegradation of commercial sugar beet production and sugar yield, or the contribution of the pathogen, the hyphae of the fungus absorbing and retaining some of the amino acids for the synthesis of its own proteins, may all contribute to the quantitative increase of specific amino acids in the infected tissues (Rossi et al., 2000; Weiland & Koch, 2004; Kaiser et al., 2010; Skaracis et al., 2010 and El–Mansoub, et al., 2017).

The data in Table 4 suggest that, Blight Stop treatment showed the highest reduction in juice impurities and α-amino N, followed by Trichoderma harzianum during the two successive growing seasons 2020/21 and 2021/22 compared with control treatment. B. megaterium treatment was the least effective. This might be because biological treatments have a role in decreasing disease incidence and severity, which is reflected in a decline in the impurities in juice as argued by Martin et al. (2001) and Hamden et al., 2023.

The data in Table 5 demonstrate that all tested biological treatments significantly increased total soluble solids (TSS), sucrose, and purity percentage when compared to the untreated plants. Spraying Blight Stop resulted in the largest increase in TSS, sucrose and purity percentages during both seasons, followed by T. harzianum. These results are in agreement with those reported by Rossi et al., (2000), Schmittgen, (2015) and Stevens, (2017), who found that BCAs efficiently controlled Cercospora leaf spot, and simultaneously promoted the plants growth (Chen et al., 2022).

The data in Table 6 showe the largest increase the root, top and sugar yield was observed when sugar beet plants were sprayed with Blight Stop twice, followed by T. harzianum compared to the other treatments. The obtained results are in agreement with those reported by Cioni et al. (2004); Stefania et al. (2008), Farahat (2018) and Morsy et al. (2022), who reported that bioagents may increase the nutrients and essential elements required to improve photosynthesis in the host plants in order to reduce the negative effects of the pathogen and increase root and sugar yields. Results in Tables 4,5 and 6 show that, in comparison to the untreated control all tested treatments recorded high values for the quality traits (sucrose and purity percentages), root, top and sugar yields of sugar beet, and lowest juice impurities (K, Na and α-amino N%). This discovery may be attributed to a decline in impurities, such as sodium, potassium, and alpha amino-N levels, as well as a decline in disease incidence and severity, which has an impact on root production and sugar content.

Conclusion

Our finding with the commercial biocides—Blight Stop (Trichoderma harzianum), Bio Zeid (T. album), Root Guard (Bacillus subtilis), and Bio ARC (B. megaterium)—as well as, four biocontrol agents (T. harzianum, T. album, B. subtilis, and B. megaterium) sprayed at the recommended doses with 15 days between sprays show a significant reduction in Cercospora beticola, total amino acid and juice impurities in comparison to control treatment. All bioagents and biocides put to the test showed a significant increase in phenolic compounds, total chlorophyll, sucrose (%), purity (%), root, top and sugar yield quality of sugar beet during both seasons. Spraying Blight Stop was the most effective treatment followed by T. harzianum, however B. megaterium was the least effective biocide treatment compared with control treatment during the two growing seasons 2020/21 and 2021/22.