Performance efficiency of some biocontrol agents on controlling Cercospora leaf spot disease of sugar beet plants under organic agriculture system

This investigation was carried out on commercial organic field in the Fayoum Governorate of Egypt under conditions of natural infection, 2020/2021 and 2021/2022 to evaluate the effectiveness of four 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) in controling Cercospora beticola Sacc, the main causal of Cercospra leaf spot (CLS) disease on sugar beet in an organic farming systems. All biocontrol agents and commercial biocides sprayed at the recommended dose in a two spray regime with 15 days between sprays, were significantly reduced Cercospora beticola, total amino acid and juice impurities (K, Na and α-a N %) in comparison to control treatment. All bioagents and biocides put to the test resulted in a significant rise in phenolic compound values, 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. B. megaterium was the least effective biocide treatment compared with the control treatment during the two growing seasons.


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 andAmer et al., 2019).
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., 2008Shahraki et al., 2008;Jacobsen, 2010 andDerbalah 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).
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.

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./50Lit.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:- 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 andMorsy 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.

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: 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 m 2 , 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 15 th 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: 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: 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).

��
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).

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).

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

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.

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).
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.
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.

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.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",  et al., 2004 andDerbalah 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, andSharma, 2015).Trichoderma spp.can also create antifungal compounds such as trichodermin, alpha-1,3-glucanase, betaglucosidase, and endo chitinase (Galletti et al., 2008, Stefania et al., 2008and 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 Cercosporinrelated toxins, which have a negative impact on plant health and photosynthesis (Scholes &Rolfe, 2009 andEl-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 andChaerle 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 andEl-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 Vol.: (0123456789) 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.
Author's contribution Majority contribution for the whole article belongs to the author.The author read and approved the final manuscript.Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

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

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 Vol:. (1234567890)

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 Vol.: (0123456789)

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