The influence of microbiological preparation on sugar beet infection 
by  Rhizoctonia  solani Kűhn

Suzana Kristek1 , Ivo Rešić2, Renata Bažok3,  Jurica Jović1, Ivana Varga1

1 University of J. J. Strossmayer, Faculty of Agronomy in Osijek
2 Sladorana d.d., sugar factory in Županja
3 Faculty of Agronomy in Zagreb

	Sugar beet is a highly demanding industrial plant, which requires deep soils of good pedo - physical, chemical and microbiological characteristics, as well as a proper agrotechnics. However, if all agrotechnic measures are properly conducted every year, the weather condition have great influence on the development of root rotting (Rhizoctonia solani Kűhn) of sugar beet.
	The fungi Rhizoctonia solani Kűhn is the most dangerous cause of sugar beet root rot. It  infects young sugar beet from the emergence phase during growing season, causing high losses in storage facilities where the plants are piled in pyramids until the processing in sugar factory. Intensity of the infection depends on weather conditions in the growing season (1), as well as on physicochemical and microbiological soil properties. Soils of poorer structure and water air soil regime, along with lower pH values and generally poorer qualitative and quantitative composition of microorganisms usually have significantly larger number of spores of the pathogen; consequently, the risk is higher of the infection in young sugar beet plants (2, 3, 4, 5).
	There is a growing interest aiming at application of microbiological preparation containing beneficial microorganisms that have microbicidal effect on soilborne pathogens and that influence the intensity of humification of organic matter and humus mineralization in  soils. Furthermore, as was shown by (6, 7, 8, 9), all natural resources have been put to a  maximum use by combining microorganisms' strains that exhibit no competitiveness or antagonism, but positive synergistic activity.
	The EM Aktiv is a microbiological preparation of complex activity, because it contains bacterial strains that take free nitrogen from the air, bacteria that promote processes of humification and mineralization in the soil and moreover, beneficial bacteria showing microbicidal activities against soilborne pathogen  R. solani Kűhn. For these reasons we   based our research on the hypothesis that the application of EM Aktiv could reduce sugar beet plant infection with the pathogenic fungi R. solani Kűhn. 

Materials and methods
Research related to root rot of sugar beet caused by pathogenic fungi Rhizoctonia solani Kűhn were conducted on two soil types: Eutric brown soil and Humogley (Table 1) during 2013 and 2014, accordance with scheme completely randomized block design with four replications and 12 different variants that included 2 tolerant hybrids  (Santino, breeding house Strube; Jadranka, breeding house KWS) and 2 sensitive hybrids (Fred, breeding house Strube; Terranova, breeding house KWS) to this pathogenic fungi. There were 3 different variants of application of microbiological preparations: control (1), the soil was treated pre sowing (2) and pre sowing and foliar (3) with the biofertilizer EM Aktiv (Em technology, Valpovo, Croatia), which contain benefit bacteria (Pseudomonas fluorescens Migula, Bacillus megaterium de Bary, Bacillus subtilis (Ehrenberg) Cohn, lactic acid bacteria) that show extremely antagonistic action against pathogenic fungi R. solani  Kűhn.
Table 1.  Soil properties.

Investigated parameter	Soil	
0 – 30 cm	Eutric brown soil	Humogley	
pH (H2O)	6.62	7.49	
pH (KCl)	5.79	6.65	
Humus (%)	1.88	2.90	
P (mg/ 100 g soil)	21.43	25.38	
K (mg/ 100 g soil)	20.07	24.76	

Microbiological preparation EM Aktiv was applied pre sowing (applied to the soil and incorporated by seed harrow) at the rate of 40 l/ha  and  applied pre sowing at the rate of 30 l/ha with a foliar application of 10 l/ha (in the phase of closing of rows).
The percentage of the plants infected with pathogenic fungi R. solani Kűhn and percentage of decayed plants were determinated in 2 - 4 true leaves phase. After the harvest in the middle of October, root yield (t/ha), sugar content (%) and sugar yield (t/ha) were determined.
Results were processed by modern statistical methods (ANOVA) using the computer program StatSoft Inc. STATISTICA [9] - data analysis software system (10).

Results and discussion

Infected and decayed plants
The least number of infected and decayed plants in the 2 – 4  true leaves phase, on both soil types, and in two years of investigation (Table 2) was obtained with the variants 2 and 3, although in most cases there was no statistically significant differences between these variants. 

Table 2. The average (2013 and 2014) percentage of the infected and decayed plants as a consequence of R. solani  Kűhn infection in the 2–4 true leaves stage.

Hybrid	
Variants	Eutric brown soil	Humogley	Average
 of 
A
(%)	Average 
of 
B
(%)	
		A   
 (%)	B
(%)	A
(%)	B
(%)			

Tolerant
on 
R. solani	
Santino	1	23.33	15.91	21.56	14.07	22.45	14.99	
		2	8.97	6.08	8.40	5.02	8.69	5.55	
		3	9.17	6,46	8.11	5.14	8.64	5.80	
	
Jadranka	1	21.87	15.06	21.02	14.72	21.45	14.89	
		2	7.88	5.56	7.56	5.13	7.72	5.35	
		3	7.94	5.10**	7.28*	4.73**	7.61	4.92	

Sensitive
on 
R. solani	

Fred	1	31.15	21.11	29.04	18.95	30.10	20.03	
		2	13.73	9.11	10.63	6.81	12.18	7.96	
		3	12.90	8.89	10.81	7.11	11.86	8.00	
	
Terranova	1	26.44	19.58	25.03	16.79	25.74	18.19	
		2	10.96	7.44	9.81	6.05	10.39	6.75	
		3	11.14	7.50	9.96	6.03	10.55	6.77	
LSD0.05
LSD0.01	0.27
0.41          	0.13
0.24	0.22
0.37	0.11
0.19	0.24
0.40	0.11
0.21	

Variants: A – infected plants; B - decayed plants; 1 – control; 2 – soil; 3 – soil + plant

The obtained results are in agreement with those of  (9) who reported that the plants inoculated with bacteria P. fluorescens Migula were characterized with rapid initial growth that enabled fast undergoing through the most vulnerable phase when the damage of the pathogen was the most pronounced. Due to the pronounced antagonism of the bacterium to the root decay agent of sugar beet root rotting fungi R. solani Kűhn, high survival percentage of inoculated plants was obtained in comparison to the non-inoculated ones and decrease in the damage of infected plants, which consequently, influenced the sugar beet root yield.

Root yield
On both soil types and in both investigation years the highest average root yield values were obtained with the variant 2 and 3 (Table 3), although in almost all hybrids there were no statistically significant different between this two variants. On Eutric brown soil sensitive hybrid Terranova has achieved a higher sugar beet root yield in relation to other experimental hybrids.

Table 3. The average (2013 and 2014) sugar beet root yield (t/ha).

Hybrid	
Variants	
Eutric brown soil	Humogley	Average

Tolerant
on 
R. solani	
Santino	1	66.02	69.56	67.79	
		2	71.60	72.83	72.22	
		3	71.45	73.25	72.35	
	
Jadranka	1	68.05	71.32	69.69	
		2	72.39	75.16	73.78	
		3	71.96	74.56	73.26	

Sensitive
on 
R. solani	
Fred	1	56.62	61.17	58.90	
		2	70.44	72.11	71.28	
		3	71.26	72.93	72.10	
	
Terranova	1	58.03	62.85	60.44	
		2	73.10	74.09	73.60	
		3	72.03	74.60	73.32	
LSD0.05    
LSD0.01                                                	0.92
1.74	0.79
1.58	0.83
1.63	

Variants: 1 – control; 2 – soil; 3 – soil + plant

P. fluorescens Migula produces cyclic lipopeptides indicating antibiotic properties against pathogenic fungi on the sugar beet roots (11, 12). In this way infection possibility is reduced i.e. less plants become infected and infection intensity is weaker. Apart from that, the pronounced antagonism, due to antibiotic substances production, (13) reported that P. fluorescens Migula is direct competitor to these fungi for both nutrients and infective spots on the root surface (4, 14).
P. fluorescens Migula shows distinguished biosurfactant properties (11) and improves general plants condition resulting in arable crops yield and quality. As the results of their researches authors stated that P.  fluorescens Migula stimulated nutrients uptake (15), increases photosynthesis intensity, as well as solubility of phosphorus inorganic forms. Moreover, this bacteria improves plants vigor and increases yield by 15-20% (4).

Sugar content 
The highest sugar content value in both investigation years and on both soil types was also obtained  in variants 2 and 3 (Table 4).
Similar results were reported in the other studies (16, 2, 6).

Table 4. The average (2013 and 2014) sugar content (%).

Hybrid	
Variants	
Eutric brown soil	Humogley	Average

Tolerant
on 
R. solani	
Santino	1	13.68	14.05	13.87	
		2	14.76	15.26	15.01	
		3	14.01	15.45	14.73	
	
Jadranka	1	13.95	14.36	14.16	
		2	14.90	15.60	15.25	
		3	14.74	15.48	15.11	

Sensitive
on 
R. solani	
Fred	1	12.77	12.96	12.87	
		2	14.20	14.55	14.38	
		3	14.39	14.78	14.59	
	
Terranova	1	13.02	13.45	13.24	
		2	14.75	15.07	14.91	
		3	14.68	14.98	14.83	
LSD0.05    
LSD0.01                                                	0.144
0.250	0.180
0.311	0.158
0.279	

Variants: 1 – control; 2 – soil; 3 – soil + plant

Sugar yield
The highest sugar yield on both soil types in two years of investigation was obtained with the variants 2 and 3 (Table 5), but in most cases there were no statistically significant differences between them. 
Table 5. The average (2013 and 2014) sugar yield (t/ha).

Hybrid	
Variants	
Eutric brown soil	Humogley	Average

Tolerant
on 
R. solani	
Santino	1	8.89	9.51	9.20	
		2	9.79	10.58	10.19	
		3	9.61	10.89	10.25	
	
Jadranka	1	9.04	9.98	9.51	
		2	9.81	10.75	10.28	
		3	9.73	10.56	10.15	

Sensitive
on 
R. solani	
Fred	1	7.48	8.96	8.22	
		2	9.12	10.04	9.58	
		3	9.05	10.11	9.58	
	
Terranova	1	8.01	9.13	8.57	
		2	9.63	10.57	10.10	
		3	9.49	10.48	9.99	
LSD0.05    
LSD0.01                                                	0.088
0.142	0.096
0.167	0.091
0.153	

Variants: 1 – control; 2 – soil; 3 – soil + plant

Both variants of microbiological preparation application increase sugar yield on average by 1.14 t/ha as compared to control.
Sugar yield was very  significantly  positively correlated  with  root  yield    (r = 0.944; p < 0.01) and sugar content (r = 0.961; p < 0.01).

Conclusion
Based on this investigations conducted the following conclusions can be made:
-	The best results in all investigated parameters were obtained by application of microbiological preparations;
-	In most cases there were no statistically significant differences between the 2 and 3  variants;
-	With reference to the fact that pathogenic bacteria rapidly develop resistance against chemical fungicides which are in varying degrees exposed to washing out and causing eutrophication of underground waters, we can consider application of biocontrol agents in soil as an acceptable option from economic and environmental point of view.

Abstract
In the sugar beet production, depending on climatic conditions, the level of infestation of the soil pathogens and agrotechnology, the problem of sugar beet root rot was appeared. Of pathogens which causes root rot of sugar beet, most damage is caused by the fungi R. solani Kűhn.  Taking into consideration the chemical and microbiological properties of the soil, the experiment was conducted on two soil types: Eutric brown soil and Humogley.
The experiment was set up in 2013 according to the scheme completely randomized block design with four replications and 12 different variants, which included two tolerant and two sensitive hybrids to this pathogenic fungi, and three different application of microbiological preparation:  control (1), the soil was treated pre sowing (2) and pre sowing and foliar (3) with the biofertilizer EM Aktiv, which contain benefit bacteria (Pseudomonas fluorescens Migula, Bacillus megaterium de Bary, Bacillus subtilis (Ehrenberg) Cohn, lactic acid bacteria)  that show extremely antagonistic action against pathogenic fungi R. solani  Kűhn. In the variant 2 microbiological preparation EM Aktiv was applied pre sowing (applied to the soil and incorporated  by seed harrow) at the rate of 40 l/ha and  in the variant 3  it was applied pre sowing at the rate of 30 l/ha with a foliar application of 10 l/ha (in the phase of closing of rows).
The percentage of the plants infected with pathogenic fungi R. solani Kűhn and percentage of decayed plants were stipulated in 2 - 4 true leaves phase. After harvest of sugar beet in the middle of October root yield (t/ha), sugar content (%) and sugar yield (t/ha) were determined.

Keywords: sugar beet, Rhizoctonia solani, microbiological preparation, yield, root quality

Acknowledgment: The presented results are part of the investigation project IPA:  „Enhancement of collaboration between science, industry and farmers: Technology transfer for integrated pest management (IPM) in sugar beet as the way to improve farmer’s income and reduce pesticide use” (IPA 2007/HR/16IPO/001-040511).
References
1. Dircks, C., Behn, A., Varrelmann, M.: Optimization of sugar beet Rhizoctonia solani resistence test in field trials by irrigation and fleece cover. Institute of Sugar Beet Research (2011), Holtenser Landstr. 77, Göttingen, Germany..
2. Kristek, S., Kristek, A., Pospišil, M., Glavaš Tokić, R., Ćosić, J.: Influence of bacterium Pseudomonas fluorescens on the pathogen of root rot Rhizoctonia solani, storage period and elements of sugarbeet yield and quality. Zuckerindustrie, 132, 2007 (7), s. 568-575.
3. Kühn, J., Rippel, R.,  Schmidhalter, U.: Abiotic soil properties and the occurrence of Rhizoctonia crown and root rot in sugar beet. Journal of Plant Nutrition and Soil Science, 172, 2009, s. 661 - 668.
4. Sanchez, C:  Symbiosis: Establishing the roots of a relationship. Nature Rev. Microbiology, 9, 2011, s. 629 – 630.
5. Bittner, V.: Organisms harmful to sugar-beets - Leaf blots on sugar-beets of fungal origin. Listy cukrovarnicke reparske, 115, 1999 (3), s. 78-79.
6. Kristek, S., Kristek, A.,  Guberac, V., Glavaš Tokić, R., Pavlović H.: Influence of Sugar Beet Treatment with Pseudomonas fluorescens and Low Fungicide Doses on Infection with Pythium and Plant Yield and Quality. Journal of Phytopathology, 154, 2006, s. 622-625.
7. Sorensen, D., Nielsen, T.H., Christophersen, C., Sørensen, J. and M. Gajhede:            Cyclic lipoundecapeptide amphisin from Pseudomonas sp. Strain Dss73. Acta Crystallogr. Sect. C Cryst. Struct. Commun. 57, 2001, s. 1123 – 1124.
8. Thrane, C., Nielsen, T.H., Nielsen, M.N., Sorensen, J., Olsson, S.: Viscosinamide – producing Pseudomonas fluorescens DR54 exerts a biocontrol effect on Pythium ultimum in sugar beet rhizosphere. FEMS Microbiol Ecol., 33, 2000, s. 139-146.
9. Whipps, J.M.: Microbial interactions and biocontrol in the rhizosphere. Journal of Experimental Botany, 52, 2001, s. 487 – 511.
10. www.statsoft.com
11. Nielsen, T.H., Christophersen, C.,  Anthoni,  U., Sørensen, J.:  Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54. J. Appl. Microbiol. 87, 1999, s. 80-90.
12. Koch, B., Nielsen,  T. H., Sørensen, D., Andersen, J. B., Christophersen, C., Molin, S., Giskov, M., Sørensen, J., Nybroe, O.:  Lipopeptide production in Pseudomonas spp. strain Dss73 is regulated by components of sugar beet seed exudates via the Gac two – component regulatory system. Appl. Environ. Microbiol. 68, 2002, s. 4509-4516.
13. Elad, Y., Baker, R.: Influence of trace amounts of cations and siderophores producing pseudomonads on chlamydospore germination of Fusarium oxysporum. Phytophatology, 75, 1985, s. 1047-1052.
14. Neu,  T.R.: Significance of bacterial surface – active compounds in interaction of bacteria with interfaces. Microbiol. Rev. 60, 1996, s. 151-166.
15. Duijff, B. J., Meijer, J. W., Bakker, P. A. H. M., Schippers, B.: Siderophore mediated competition for iron and induced disease resistance in the suppression of Fusarium wilt of carnation by fluorescent Pseudomonas spp. Neth. J. Plant Pathol., 99, 1993, s. 277-291.
16. Pedersen, H.C., Weiergang,  I., Pontoppidan,  M.M., Jørgensen, L., Svingel, A.:  Danisco Seed, Seed Technology Dept., (2002). Hoejbygaardvej 31, DK – 4960 Holeby, Denmark.

Contact address:

                   Ph.D. Suzana Kristek, full professor
                   University of J. J. Strossmayer
                   Faculty of Agriculture
                   Kralja P. Svačića 1d
                   31000 Osijek
                   Republic of Croatia
                   e-mail: skristek@pfos.hr