New Approach to Control Sclerotium rolfsii induced sugar Beet Root Rots Disease by Trichoderma with improved sucrose Contents

in egypt, sugar beet (Beta vulgaris L.) has become a major sugar manufacturing plant in latest years. It is recognized that sugar beet damaged by different pathogens, including root rot disease caused by Sclerotium rollsii, in terms of quantity and quality. The aim of the current study was to control the disease of the root rot sugar beet and determine the sucrose content during two successive cropping seasons. Trichoderma harzianum kj831197 produced bglucanase enzymes that play a key role in fungal disease biocontrol. Twenty two bioactive isolates were tested for the activity of bglucanases, ten of which are Trichoderma spp strains. Sclerotium rolfsii radial growth has been suppressed with efficiency ranging from 77.77 to 91.11% in dual culture technique. The Vitavax200 fungicide increased control of the disease under greenhouse conditions followed by a combination of bglucanase enzyme with Trichoderma harzianum kj831197 spore suspension. The use of bglucanase enzyme mixed with Trichoderma harizianum kj831197 cells leads to an increase above other treatments in the total soluble solid and sucrose content of the sugar beet. Despite the fungicide Vitavax200, the overall soluble solid and sucrose content were significantly affected by disease control but the sugar beets yield was lowered.

genus Trichoderma produces different kinds of enzymes which play a major role in biocontrol activity like degradation of the cell wall, tolerance to biotic or a biotic stresses, hyphal growth etc.
Our previous data (under publication) in medium optimization giving rise of maximal glucanase enzyme production of 10 folds higher than the initial medium owing to the optimization of growth parameters. The optimal condition for enzyme production was highly increased by xylose as C-source 0.5%, 0.1mM tween-80 and 2mM galactose added as an inducer, pH 5.5, temp. 37 o C, agitation 200 rpm, nitrogen source was malt extract at a conc. of 1 %. Through large scale production of bglucanase enzyme of the optimized medium in bench-scale bioreactor will be conducted. This work conducted for using the results to complete the picture by making a practical application in greenhouse experiment. The other main objective is the application of highly active b-glucanase enzyme and Trichoderma harizianum kj831197 as an eco-friendly biopesticide alternative to chemical pesticides.

MAteRiAls AND MethODs Chemicals
Lichenan as glucanase substrate (purchased from Sigma-Aldrich), Dinitrosalicylic Acid (DNSA), other chemicals were of analytical grade.

Bioreactor
For large scale production of b-glucanase batch cultivation was achieved in a 3L bench-top bioreactor (Bioflow III, New Brunswick, NJ, USA) supplied with two 6-bladed disc-turbine impeller and four baffles, and joined to a digital managing system. The process was computerized through the AFS BioCommand multi-process management system, computer-assisted data processing system and the limits set by mechanical filling of weak acid or base for the physical properties at a temperature of 30 ° C and pH 6 were established. Originally compressed air was supplied by a sterile filter to 1.0 VVM. The dispersed oxygen level remained above 20% and can then be manually adjusted in addition to the agitation speed (200rpm). The dissolved oxygen level was above 20%. The dissolved oxygen level ad pH values were determined online with Mettler Toledo electrodes and antifoams A (Sigma), for the elimination of foaming. Inoculums of Trichoderma harizianum kj831197 injected as spore suspension of 10% in the optimized medium used in fermentor which was xylose (C-source) 0.5%, 0.1mM tween-80 and 2mM galactose added as an inducer, pH 5.5, temp. 37 o C, agitation 200 rpm, nitrogen source was malt extract at a conc. of 1 %.

Experimental design
The present study was performed in two positions under laboratory and greenhouse circumstances. Sclerotium rolfsii the causal agent of sugar beet root rots disease was isolated from infected beet plants samples and bioagents isolates were detached from rhizosphere soil specimens. The bioagent showing the highest glucanase activity production was chosen for identification and further analyses. Identification has been made morphologically by the aid of a microscope and later by molecular means.

Estimation of the bioagents organisms for glucanase production
Glucanase enzyme was quantitative estimated using the method of Miller 10 , in which 1g of DNSA dissolved in 50 ml distilled water, 30g of sodium-potassium tartrate slowly add. Stirring the mixture till complete dissociation, 20 ml of 2N NaOH added. All the above steps must be carried out in the dark bottle enclosed in aluminum foil as DNSA is light-sensitive solution, Store the bottle at room temperature and it must freshly prepare.

Glucanase enzyme activity assay
From Trichoderma harizianum kj831197 broth culture, 1 ml culture centrifuged at 10,000 rpm for 10min, the supernatant constitute the extracellular enzyme production. For enzyme assay, prepare 0.5% lichenan as the substrate of glucanase enzyme in 0.1M phosphate buffer pH (4.5-5), enzyme assay mixture consisted of 0.5 ml of the prepared substrate mixed with 0.5 ml of the supernatant containing the enzyme in test tubes, incubate the tubes at 34 o C in water bath for 20 min, mix gently from time to time, stop the reaction by adding 1 ml of DNSA, boil the tubes for 10 min at 100 o C, for blank tubes all previous contents added replacing the enzyme by 0.5 ml of distilled water or the buffer, measure the absorbance at 540 nm against the blank. Glucanase activity was defined as 1 micromole of glucose released per minute under assay condition and this can be calculated according to Kumala et al. 11 .

Efficacy of the tested bio-control agent against sclerotium rolfsii under green-house conditions
Greenhouse study has been planned to estimate several treatments for Sclerotium rolfsii; i.e. culture of the bio-control agent, fungicide (Vetavax200) and clove (Syzygium aromaticum) in controlling sugar beet root-rotting. During the 2015/2016 and 2016/2017, the test was conducted.
Two techniques of implementation have been used, i.e. bio-control agents and seed treatment:

Bio-formulation of bio-control agents
In this experiment, Talc powder was used as a carrier substance for bio-formulation of Trichoderma biomass. Talc powder (300 meshes, white color) 2Kg, carboxy methylcellulose/Gum Arabic powder 10g and Gypsum powder 2Kg prepared. Construction of powder formulation was brought by running stationary culture approach. The biomass from the 15day culture of Trichoderma prepared in flasks was utilized for the establishment of the formulation. The biomass along with the medium in conical flasks was mixed with a carrier in the rate of 1:2. The mixture was air-dried in shade for 3 to 4 day and blended to have an owing powder to which 0.5% sucker (CMC/ Gum Arabic) was added. The formulation therefore developed was packed in white polythene bags at room condition and in the refrigerator (4°C). The population evaluation was made at periodic intervals. seed treatment Sugar beet seed (C.V. Kwamera) the extremely sensitive variety to root-rots was applied in the performing study. Seeds were carefully cleaned by tap water and dried in the dark previous to usage. According to Abd-El-Moity and Shatla 12 , Trichoderma harzianum kj831197 was cultivated in conical flasks (250ml) containing autoclaved 100 ml molasses yeast extract broth (10g molasses, 1.6g yeast extract and 990 ml distilled water) at laboratory condition (18-20 o C) in the dark for 14 days. After the pass-by of the study time, the mature fungal growth was blended and refined applying filter sheet and the filtrate was gathered to apply in treating seeds. Dressed seeds with Vitavax 200 (Vitavaxtriram WP) at the approved dose (30g/Kg) were accepted as a Journal of Pure and Applied Microbiology control in the greenhouse study also seed dressed with glucanase enzyme of Trichoderma and treated seeds with clove oil at a dose (25,000ppm) were tested.

Inoculums of sclerotium rolfsii
The pathogen was developed on the sand and wheat (2:3 w/w) into glass bottles for two weeks at 27 o C. After the establishment time, the cultivated fungus was adopted in invading clay soil at the rate of 2% of soil weight. The soil was, dispersed into clay pots, rinsed with tap water and allowed for one week to enable the infection to settle itself before sowing. Pots were, implanted with seeds (10 seeds/ pot), moistened and enriched as frequent. Four replicates/ treatment were valued and pots buried with untreated seeds served as control.

Disease assessments
Pre-and post-emergence damping off was scored after 15 and 45 days of planting, respectively, according to El-Shafey et al. 13 as follows: Pre-emergence damping-off (%) = (No. of non-emerged seed/Total No. of seeds sown) x 100. Post-emergence damping-off (%) = (No. of wilted plant /Total No. of the emerged plant) x 100. Root rot severity was scored 150 days after planting based on with the following ratings: 1no internal or external browning. 2-No internal browning, with superficial lesions of ≤ 25% on tap root. 3-Slight internal browning with < 25 -≤ 50% surface covered with cankers. 4-Moderate internal browning with <50 -≤ 75% cankers. 5-Severe internal and external browning, i.e. rot covered <75% of the root surface.
Where: n= No. of plants in each numerical rate (r0….r4). N= Total No. of plants multiplied by the maximum numerical rate (4).

Assessment of the yield components
At harvest, 150 days after planting, the yield was determined as weight of sugar beetroots (g), samples of the roots (4 replicate) of each cultivar were immediately transferred to the laboratory for total soluble solids and sucrose content assessments.

Assessment of sucrose and total soluble solids (tss)
Sucrose % and total soluble solids (TSS %) was determined at harvest. The (TSS %) was determined in fresh roots using hand Refractometer 14 . While, sucrose percentage was estimated by adding 26 g from the minced root to 177 ml of lead acetate (50 g/liter of distilled water), shacked for 5 minutes and filtered. The filtered solution was measured by Saccharometer as mentioned by Le-Docte 15 .

Data analysis
The tests were conducted with three replicates. Data were subjected to analysis of variance (ANOVA) and least significance difference (LSD) used to compare the means for all the variables within the experiment at (P=0.05) 16

ResUlts
bglucanase enzyme synthesis increased rapidly during the first 20 hours of cultivation, though the highest levels were reached after 60 hrs. The changes in bglucanase enzyme content during the growth of the selected bioagent reflect the changes in the growth rate during the different phases of the growth. The level is minimum when cells are in the lag phase, do not divide and their growth rate is low. As the cells start to divide, their number and the bglucanase enzyme content increase continuously as the growth rate reached its maximum value. This corresponds to the exponential phase of the culture, after which the culture enters the declaration phase. During this phase cell population continues to death, while the growth rate decreases continuously. During these two phases, the carbon sources become exhausted, and cells convert their products reserve to a form utilizable for their metabolism and division. Hence, paramylon level in the culture simultaneously decreases. The highest bglucanase enzyme concentration was reached 2000 U/ml (Fig. 1) reached in reduced time and energy only takes 60 hr in contrast to the previous results ( in shake flask) giving 1354 U/ml that takes 6 days operation.

Assessment of disease parameters
Data presented in Table 1 indicated that, the treatment with b-glucanase enzyme enhanced seedling emergence, (after 15 days from planting),  with S. rolfsii was significantly reduced due to any of treatments (Fig. 2). Data in Table 3 and (Fig. 3) Showed that the addition of glucanase enzyme only and in combination with cells of Trichoderma harzianum kj831197 increased the weight of sugar beetroots, while Syzygium aromaticum treatment ranked after them in weight of roots of sugar beet comparing to untreated control.  Table 4 showed that the effect of the metabolites of Trichoderma (glucanase enzyme) and in case of its combination with cells of Trichoderma harzianum kj831197 compared to other treatments was increased TSS and sucrose content of sugarbeet. Significant differences were found between values of TSS and sucrose content of sugarbeet as affected by combined treatments of glucanase enzyme and cells of Trichoderma harzianum kj831197. The highest reduction was detected in control with and without talc and also with Syzygium aromaticum and Vetavax fungicide treatments.

DisCUssiON
Sugar beet (Beta vulgaris L.) is considered as the second important source for sugar production following sugar cane in Egypt. Rootrot disease, caused by some common fungi, . Biocontrol components may provide the correct values for infection tolerance as an alternative for certain chemical fungicides. Only a few microorganisms were fully marketable to protect foliar crop pathogens 18 . Trichoderma harzianum (Trichodex20SP), which can be regarded as a model of natural biocontrol agent to demonstrate its impact in agricultural conditions 19 .
The formulation makes up originally of a microorganism and an additive serve as a carrier. Powder or granular inert components can be combined matrices such as rock wool and peat-based mixtures, clays, kaolin clay, montmorillonites, saponites, mica, perlites, vermiculite, talc. Support for preserving and the safety of the microbes against the impact during storage and ship should be steady and pure to set and distribute. All formulations are still requested to be efficient 20 .
Adequate volume is required with both solid and liquid formulations for effective and viable Trichoderma inocula. The fluid model is advanced with the aim of maximizing biomass output and effectiveness by regulating nutrient components, pH and temperatures as well as additional development factors that cause contamination to decrease 21 . That is what we do in our study; by comparing the obtained results in bioreactor for large scale production of bioagent and its components with those previously obtained in shake flask or solid-state fermentation at the same condition, we gained maximum enzyme activity at shake flask reached 1354 U/ml after 6 days (under publications), while at bench top fermentor it reached more than this level (2000 U/ml) with little time (60hr only). This high value of enzyme activity at bench top fermentor may be due to the use of considerable level of gas flow which equal to 1 vvm and controlled growth conditions of agitation, pH and aeration. Accordingly, results obtained in a shake flask should be taken only as preliminary indicators of the conditions necessary for successful scaling up bioagent production and must be verified in studies carried out in a fermentor. The dissolved oxygen (DO) rapidly decreases during the exponential growth phase because of the respiration of the cells. During the stationary phase (DO) levels increases probably because of a decrease in the respiration rate of Trichoderma cells. This increase of dissolved oxygen is due to the lack of substrate and is used as a signal to feed the fermentor [22][23] .
Results of this research demonstrate that Trichoderma harizianum kj8311197 produce a high quantity of b-glucanase enzyme. The b-glucanase enzyme developed by the Trichoderma has been greatly reduced the radial size of the S. rolfsii that was in agreement with Mala et al. 24 .
One of the principal processes for antagonistic action against phytopathogenic fungi was the immediate mycoparasitic action of Trichoderma species 25 . Trichoderma fungal species produce distinctive hydrolytic enzymes used as biocontrol agents that show a key role in cell wall deterioration. Hydrolytic enzymes include chitinase, glucanase, protease and cellulase 26 . Castillo et al. 27 found that T. longibrachiatum and T. asperellum were the most efficient species with the highest antagonist effects against Sclerotinia sclerotiorum and Sclerotium cepivorum among Mexicans Trichoderma strains.
Chakraborty et al. 28 recorded that combined therapy of Bradyrhizobium japonicum and Trichoderma harzianum markedly lowered Soya bean plant rot infection. The proposed mechanisms of Trichoderma as antagonistic bioagent were identified: (1) powerful mineral fight; (2) antibiotic development through helpful microorganisms; (3) successful predation against pathogens by secretion of hydrolytic enzymes. These fungi can, therefore, stimulate root development, regulate deleterious pathogenic microflora, the decay of harmful microflora producing toxic metabolites and regulate the root pathogens immediately. Today, in several agricultural provinces more than 50 different agricultural products depending on Trichoderma are applicable and result in higher plant revenues 29 .
Recently, biological control agents (BCAs) based on Ttrichoderma has grown to approximately 60% of all fungal BCAs. T. Harzianum has lately been used as an effective element in various commercially available biopesticides [30][31][32] . Marketing of biocontrol agents are a multi-step method including microorganism isolation, selection of the best antagonistic isolate in laboratory circumstances and field environments, mass processing, formulations, production and compatibility 4 .
Addition of glucanase enzyme of Trichoderma harzianum kj831197 to cells of Trichoderma harzianum kj831197 increased the yield components i.e. the average of roots weight while both of Vetavax fungicide and Syzygium aromaticum reduced the yield components. Our early work demonstrated that Trichoderma sp. did indeed have the ability to control plant diseases. Obviously, the level of efficacy and the reliability of simple approaches to biocontrol gave results that were substantially equal to that of commercial fungicides. Glucanase enzyme of Trichoderma harzianum kj831197, enhance plant growth and productivity. Aly and Hussein 33 discovered that sugar beet crops already have T. harzianum treatment giving rise high proportion of the new and dry weight of the leaves and sugar beet's roots when contrasted to control. Our results recorded significant differences between values of total soluble solids (TSS), sucrose contents of sugar beet affected by combined b-glucanase enzyme and cells of Trichoderma harzianum kj831197. All of the treatments showed an increase in % of TSS and sucrose contents except control treatment, which showed a significant reduction. The addition of Vetavax200 fungicide caused a reduction in the total soluble solid % (TSS) and sucrose contents Also, the use of Syzygium aromaticum individually showed low values of percentage of TSS and sucrose contents. The lowest percentages of TSS and sucrose contents were obtained from treatments of S. rolfsii only. These results in agreement with Aly and Hussein 33 , they reported that sugar beet crops handled with Trichoderma were revealed to be sucrose levels (%) greatly above the other medications or treatments. In addition, the sugar beet crops inoculated with Trichoderma was significantly improved in purity (%). Total soluble solids % was significantly enhanced when handling beet plants of Rhizoctonia and Trichoderma in contrast with the control of the 1st and 2nd growing seasons. Dovil‫כ‬ et al. 34 observed the small impact of fungicides on sugar beet yield and sucrose quantity. The Trichoderma sp. can grow in a wide range of habitats and this is achieved by evolved diversified metabolic enzymes and secondary metabolites. Production of commercially important enzymes such as amylases, cellulases, 1-3 bglucanases, and chitinases were extensively studied and this technology is continuously being updated [35][36][37] .This unique methods for using hydrolytic enzymes as a biocontrol agent have been raised the agricultural production in all horticultural sectors and immediately find environmentally friendly solutions to overcome problems caused by the standard chemical methods of plant protection.
Current attempts are now underway of increasing the output of hydrolytic enzymes (Glucanase) by fermenting agricultural waste materials and by optimizing growth conditions.