6 Inoculation of Sugar Beet Seed with Bacteria P . fluorescens , B . subtilis and B . megaterium – Chemical Fungicides Alternative

The most frequent decay pathogens on sugar beet seedlings are the fungi Aphanomyces cochlioides, Pythium ultimum and Pythium debarianum, as well as Rhizoctonia solani. In the growing season plant decay is most frequently caused by the fungi Rhizoctonia solani, Fusarium spp., and by the bacteria Erwinia spp. From the root rot pathogens most damage is inflicted by Rhizoctonia solani. Besides weather conditions that influence this type of root decay, the inadequate soil management – irregular soil cultivation resulting in damaged soil structure and rise in soil acidity – is a contributory factor.


Introduction
During the process of sugar beet cultivation, depending on weather conditions, the degree of soil infection with pathogenic microorganisms, and the application of soil management, the presence of sugar beet root rot is frequently observed.Pathogens are numerous, inflicting serious damage.
According to the occurrence time of root rot symptoms, the pathogens can be divided into three major groups: pathogens occurring during the emergence phase, pathogens occurring in the growing season, and ones that cause sugar beet root rot during the period of storage.
The most frequent decay pathogens on sugar beet seedlings are the fungi Aphanomyces cochlioides, Pythium ultimum and Pythium debarianum, as well as Rhizoctonia solani.In the growing season plant decay is most frequently caused by the fungi Rhizoctonia solani, Fusarium spp., and by the bacteria Erwinia spp.From the root rot pathogens most damage is inflicted by Rhizoctonia solani.Besides weather conditions that influence this type of root decay, the inadequate soil management -irregular soil cultivation resulting in damaged soil structure and rise in soil acidity -is a contributory factor.
Apart from the root pathogens occurring during cultivation in the field, certain pathogens cause sugar beet decay in the period of storage, and before processing.Storage conditions, as well as plant damages caused when digging are contributory factors in the occurrence of fungal diseases.
The most frequent pathogenic microorganisms observed are fungi, such as Rhizoctonia solani, Botrytis cinerea, Penicillium spp., Aspergillus spp.... The pathogenic fungus Rhizoctonia solani, if compared with all other sugar beet root rot pathogens, is able to infect the plant from the emergence phase, during growing season, until the process of sugar beet storage.
Fungicidal treatment of seeds, use of tolerant hybrids, or proper soil management does not ensure full plant protection against this pathogen.Moreover, heavily infected soils and weather conditions favourable for development of the pathogen, contribute to the infection to higher or lower degree.
Agronomists are faced with a growing problem of evolving resistance of soil pathogens to chemical fungicides.Also, there is an increasing sensitivity to the importance of health food production and rising awareness for environmental protection.Namely, all pesticides which are not biodegraded in the soil are being washed out in deeper layers causing eutrophication of underground waters.Therefore, natural resources and processes in the soil are widely used as an alternative to chemical fungicides.
Seed inoculation with the bacteria showing antagonistic activity against pathogenic fungi such as Rhizoctonia solani, Pythium ultimum, P. debarianum, Phoma betae and Aphanomyces cochlioides account for an alternative to chemical fungicides and an option of solving problem of disease control, not only in sugar beet, but in other, mainly vegetable crops.Many authors (Koch et al., 2002;Sorensen et al., 2001;Thrane et al., 2000;Whipps, 2001) have studied control of these pathogens by the beneficial bacterium Pseudomonas fluorescens which shows pronounced antagonistic activity against the fungi.Similarly, results of their studies proved positive effect of the bacterium Bacillus megaterium against the same root decay agents of sugar beet (Asaka & Shoda, 1996;Choudhary & Johri, 2009;Thrane et al., 2000).However, some authors proved antagonistic effect of certain beneficial fungi and yeast (Trichoderma harzianum, Candida valida, Rhodotula glutinis, …) against these pathogenic fungi (Abada, 1994;El-Tarabily & Sivasithamparam, 2006).
Pseudomonas fluorescens belongs to rod-shaped, asporogenous, gram-negative bacteria that as saprophytes prevalent in soil and water belong to a group of soil microorganisms crucial to the process of soil denitrification.
Bacillus subtilis and Bacillus megaterium form symbiotic relationship with the root system of field crops, and like P. fluorescens show antagonistic activity against these pathogens.In this study we have investigated synergistic effect of the bacteria in order to achieve as better results as possible.
Bacillus is a genus of Gram-positive rod-shaped bacteria and a member of the division Firmicutes.Bacillus species can be obligate aerobes or facultative anaerobes.The species Bacillus subtilis and Bacillus megaterium participate in nitrogen cycle, and in the soil processes of organic matter humification and mineralization of humus, especially in mineralization of phosphorus and potassium converting them into plant-accessible forms.These bacteria belong to biological control agents, i. e. they produce antimicrobial substances that exhibit antagonistic activity against soilborne pathogens, agents of diseases in arable crops (Sorensen et al., 2001).
Therefore, seed inoculation with bacteria showing antagonism against pathogenic fungi is an acceptable alternative to chemical pesticide application (Andersen et al., 2003).
Moreover, considering heavily infected soils with the pathogen R. solani and the fact that beneficial bacteria Pseudomonas fluorescens, B. subtilis and B. megaterium are not sensitive to a low dose fungicide (Pedersen et al., 2002), it is possible to treat the seeds combining low doses of the chemical agents aiming to control growth and reproduction of the pathogenic fungi, which in consequence, produce positive effect on all the parameters of sugar beet yield and quality.

Materials and methods
The experiment was set up on two types of the soil: Mollic Gleysols (FAO, 1998) and Eutric Cambisols.
On Mollic Gleysols soil type in the period of 2005 -2009 pathogenic fungus R. solani (FAO, 1998)  Hybrid of Belinda (KWS) sugar beet was used in the sowing.In the three trial years the sowing was conducted in the second decade of March.The row spacing was 50 cm, with 20 cm within row.Adequate plant stand was obtained with no corrections necessary.
Percentage of the plants infected with pathogenic fungi R. solani and P. debarianum as well as percentage of decayed plants was stipulated in 2 -4 true leaves phase.The sugar beet digging conducted in the middle of October was followed by determination of root yield (t/ha), sugar content (%), sugar in molasses (%) and sugar yield (t/ha).
Weather conditions appeared to affect sugar beet growth.Data obtained from the Agrometeorological bureau in Osijek, Croatia (Table 2) were used in weather analysis.
Weather conditions in 2007 were characterized by the increase in air temperature of more than 1 0 C above multiyear average.Precipitation rates in the growing season measured 74% out of the average, with rainfall deficiency in June and July, and excessive rainfall in September and October which was unfavourable for sugar beet growth.2008 and 2009 were also characterized by higher air temperatures in comparison with multiyear average, which was unfavourable for sugar beet growth due to the values significantly (2.5 o C in 2008; and 3.0 o C in 2009) above the optimum.Precipitation rates in the growing season 2008 were level with the multiyear average, with rainfall deficiency in August and excessive rainfall in September.Considering precipitation amount in the growing season (April -September), 2009 was dry.Only 59 % out of the average precipitation rates were measured, with rainfall deficiency throughout the year except for June.Such weather conditions were favourable for sugar beet growth until July, and then followed by the unfavourable conditions until the end of the growing season.
Results were processed by modern statistical methods (ANOVA) using the computer program StatSoft Inc. ( 2001) STATISTICA (data analysis software system), version 6.

Percentage of infected and decayed plants
During the three -year research into the influence of beneficial bacteria and chemical fungicide Thiram 42-S on the intensity of infection and decay of sugar beet plants caused by the parasitic fungi Rhizoctonia solani on Mollic Gleysols, and by the parasitic fungi Pythium debarianum on Eutric Cambisols significant influence of the bacteria and the chemical fungicide was determined in all tested variants when compared with the control (Table 3).
On The results obtained are in agreement with those of Whipps (2001) who reported that the plants inoculated with bacteria P. fluorescens were characterized with rapid initial growth that enabled fast undergoing through the most vulnerable phase when the damage of the pathogen affecting was the most pronounced.Due to the pronounced antagonism of the bacterium to the root rot agent of sugar beet -fungi R. solani high survival percentage of inoculated plants was obtained in comparison with the non-inoculated ones and decrease in the damage of infected plants, which consequently, influenced the sugar beet root yield.Kristek et al. (2007) in their research results of the effects of the beneficial bacterium P. fluorescens on the pathogenic fungus R. solani recorded obvious decrease in the number of infected and decayed plants in variants treated with the bacterium P. fluorescens when compared with the control variants.They also reported significant difference in the number of infected and decayed plants between tolerant hybrids compared with the hybrids sensitive to the pathogenic fungi.With tolerant hybrids in treated variants 4.10 % smaller number of infected plants was obtained than in the same variants of sensitive hybrids.The difference in the number of decayed plants was 3.86 %.
On Eutric Cambisols in the three trial years the best results were recorded in the variant 8 -seed inoculated with P.  Similar results in examining the effect of the bacterium B. megaterium (a constituent of the applied microbiological preparation) in the control of pathogenic fungi P. ultimum and P. debarianum were reported in the studies on the same parameters by Evačić et al. (2008).
It is evident that weather conditions in the growing season of sugar beet influenced intensity of the infection by pathogenic fungi Rhizoctonia solani and Pythium debarianum.
Namely, in the second trial year precipitation rates and air temperatures were favourable for crop development which enabled faster undergoing of young sugar beet plants through the most vulnerable phase of the growth.Consequently, intensity of the infection and the number of decayed plants as the result of the pathogen attack were both decreased which was recorded in the controls.Furthermore, there was obvious difference in the number of infected and decayed plants on different types of the soil.As it did not concern the same pathogen, it could be higher sensitivity of the hybrid to Pythium debarianum than to Rhizoctonia solani.Nevertheless, pathogenic fungi Rhizoctonia solani usually cause more economic damage in the middle and eastern European countries than pathogenic fungi Pythium debarianum.Belinda hybrid was chosen due to the high sensitivity to the fungi Rhizoctonia solani.Therefore chemical and microbiological soil properties could be presumed to have great importance in plant development and resistance against the infection caused by the pathogenic fungi.Namely, Mollic Gleysols has chemical (Table 1) and microbiological (Figure 1, 2, 3, 4, 5) properties of better quality.It is the soil of neutral reaction with high portion of organic matter and large supply of phosphorus and potassium.The soil is abundant in bacteria and actinomycetes that participate in the processes of soil organic matter humification and mineralization of humus.Large portion in microbial mass is comprised of non-symbiotic nitrogen fixers and cellulolytic microorganisms.The presence of the large number of the bacteria of the Pseudomonas fluorescens species, as well as Bacillus spp., showing antagonistic activity against the soilborne pathogenic fungi is a factor responsible for smaller number of infected plants in the controls if compared with the same on Eutric Cambisols, a soil of poorer chemical and microbiological properties.Average number of infected plants was in highly significant positive correlation with average number of decayed plants (r=0.965;p<0.01).

Root yield
Sugar beet root yield depended on soil types, or on chemical and microbiological soil properties, and on trial years.
On Mollic Gleysols in the three trial years the highest average sugar beet root yield was recorded in the variant 7 -seed inoculated with P. fluorescens No 8569 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. subtilis No 2109 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. megaterium No 2894 (0.4 x 10 10 bacteria/ha).All other variants obtained statistically highly significant lower average root yield (Table 4).Average sugar beet root yield in treated variants was 80.63 t/ha, or 21.12% higher than average root yield in the control variant (66.57t/ha).
Since the variant with the strains of beneficial bacteria being applied together obtained significantly higher average root yield than the variant with the strains applied one at a time, it can be concluded that there was highly positive synergistic activity between them.
On Eutric Cambisols in the three trial years the highest average sugar beet root yield was recorded in the variant 8 -seed inoculated with P. fluorescens No 8569 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. subtilis No 2109 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. megaterium No 2894 (0.4 x 10 10 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed), though no significant difference (p > 0.01) was determined between this variant and variant 2 -Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed).All other variants obtained highly significant (p < 0.01) lower sugar beet root yield.Average sugar beet root yield in treated variants was 69.45 t/ha or 21.08 % higher than the average root yield in the control variant (57.02 t/ha).
It is evident that on the soil type of poorer microbiological properties the best results were obtained with the application of chemical fungicides.It is also obvious that between variant 3 with application of the bacteria P. fluorescens, and variant 7 with application of P. fluorescens, B. subtilis and B. megaterium no significant differences were determined.Therefore, it can be concluded that the bacteria P. fluorescens played a crucial role in the control of pathogenic fungi P. debarianum.
Significantly better results in sugar beet production by applying plant growth -promoting rhizobacteria P. fluorescens in the control of pathogenic fungi R. solani were reported by Esh & El-Kholi (2005), as well as by Kiewnick et al. (2001) in the control of fungi Rhizoctonia crown by combination of beneficial bacteria.
P. fluorescens shows distinguished biosurfactant properties (Raaijmakers et al., 2010;Ron & Rosenberg, 2001) and improves general plants condition resulting in arable crops yield and quality.As the results of their research studies some authors stated that P. fluorescens stimulate nutrients uptake (Duijff et al., 1993;Loper & Buyer, 1991), increases photosynthesis intensity (Zhang et al., 2002) as well as solubility of phosphorus inorganic forms.The aforesaid improves plants vigour and increases yield by 15-20% (Whipps, 2001).

Sugar content
This parameter was also influenced by weather conditions in the growing season of sugar beet.Namely, higher sugar content in sugar beet root (%) was recorded in the second trial year (Table 4).
On Mollic Gleysols in the three trial years the highest average sugar content in sugar beet was recorded in the variant 7 -seed inoculated with P.  Thiram,200 ml/100 kg seed) were determined.Due to the fact that variant 3, as well as variant 7-the one which obtained the best results, were not treated with a chemical fungicide, it can be left out from application in this case.
Fungicide treated seed on finishing is obviously neither sufficient nor safe measure since the fungi fast develop resistance to these pesticide active agents (Cooke et al., 1999;Durrant et al., 1998).Furthermore, fungicides are exposed to washing out, hence the possibility of underground waters eutrophication, i.e. direct danger for a human health and environment.

Sugar in molasses
Sugar content in molasses depended, respectively, on seed treated variant, hybrid properties (tolerant, sensitive), soil type tested, and trial year.
The lowest average sugar content in molasses in the three year trial (

Sugar yield
On Mollic Gleysols the highest average sugar yield in the three trial years was recorded in the variant 7 -seed inoculated with P. fluorescens No 8569 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. subtilis No 2109 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. megaterium No 2894 (0.4 x 10 10 bacteria/ha).All other variants obtained statistically highly significant lower average sugar yield.Average sugar yield in treated variants was 10.31 t/ha or 37.83% higher than average sugar yield in the control (7.48 t/ha).It is evident that in this parameter as well, the best results were obtained in the second year of the trial (Table 5).

Conclusion
Inoculation of sugar beet seed with the bacteria Pseudomonas fluorescens, Bacillus subtilis and Bacillus megaterium affected root decay agents Rhizoctonia solani and Pythium debarianum and showed highly significant influence on all the elements of sugar beet yield and quality in the three trial years.
The best results in all elements of the research on both soil types were recorded in the second trial year (2008) due to the more favourable weather conditions (air temperatures, precipitation values) in the growing season 2008 than in the dry 2007 and 2009.
Better results in all tested parameters in the three trial years were obtained on Mollic Gleysols.The reason is in chemical and microbiological properties of much better quality than on Eutric Cambisols.In Mollic Gleysols total number of the bacteria and actinomycetes is higher enabling more intensive humification of organic matter and mineralization of humus in the plant-accessible mineral compounds.Significantly higher number of beneficial bacteria in the soil (P.fluorescens, Bacillus spp.) that show antagonism against soilborne pathogens is evident.
On Mollic Gleysols the best results in all tested parameters were recorded in the variant treated with the beneficial bacteria (P.fluorescens, B. subtilis, B. megaterium), whereas all other variants obtained significantly (p<0.01)lower values.Due to the beneficial bacteria in the soil, population of pathogenic microorganisms was reduced, making application of the beneficial bacteria in the sowing period sufficient to reduce or control the infection with pathogenic fungi R. solani.Among the beneficial bacteria P. fluorescens, B. subtilis and B. megaterium highly positive synergism is evident.
On Eutric Cambisols the best results in all tested parameters were reached in the variant treated with chemical fungicide and the beneficial bacteria (P.fluorescens, B. subtilis, B. megaterium).Nevertheless, in most cases no statistically significant difference (p>0.05) was determined between this variant and the variant being treated solely with chemical fungicide.The reason is in poorer quality of chemical and microbiological soil properties which is favourable for reproduction of pathogenic fungi P. debarianum in the soil.
If necessary measures of improving chemical soil properties are to be carried out, neutralization of soil acidity in the first place, microbiological soil properties will be improved, and the beneficial soil microorganisms will increase in number.Due to the antagonism of beneficial microorganisms against soilborne plant pathogens the number of pathogenic microorganisms will be reduced as well as the infection of arable crops.In this case the application of beneficial microorganisms will reach satisfactory level without application of chemical pesticides.By introducing beneficial microorganisms microbiological soil properties will be consequently improved and number of pathogens in the forthcoming growing season will be decreasing.The reduction in the application of chemical pesticides is the matter of great importance from the both economic and ecological point of view.

Acknowledgment
I would like to show my gratitude to Ministry of Science, Education and Sports of the Republic of Croatia for financial support of our project research (079-0791843-1933).Results presented in this chapter were obtained during this project research.
Mollic Gleysols in the three trial years the best results were recorded in the variant 7seed inoculated with P. fluorescens No 8569 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. subtilis No 2109 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. megaterium No 2894 (0.4 x 10 10 bacteria/ha).All other variants of treated seeds obtained highly significant larger number (p < 0.01) of infected and decayed plants due to the attack of the parasitic fungi Rhizoctonia solani if compared with all other tested variants.Among the variants 2 -Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed), 3 -seed inoculated with P. fluorescens No 8569 (1.2 x 10 10 bacteria/ha) and 8 -seed inoculated with P. fluorescens No 8569 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. subtilis No 2109 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. megaterium No 2894 (0.4 x 10 10 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed) no significant differences were determined (p > 0.01), though the smallest number of the infected and decayed plants was recorded in the variant 8 in the three trial years.Average number of infected plants in treated variants in the three trial years was 10.86% or 58.17% lower than the three-year average in the control variants which was 25.96%.The difference in decayed plants was 74.70%.

Fig. 4 .
Fig. 1.Average number of total Bacteria in soil during three trial years

Investigated properties in a field Type of soil Layer (0 -0.3 m) Mollic Gleysols Eutric Cambisols pH (H 2 O)
was determined, whilst on Eutric Cambisols (Table1) soil type in the same period P. debarianum was determined.

(mg/ 100 g soil) 22.80 20.55 K (mg/ 100 g soil) 23
0.4 x 10 10 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed), though no significant difference (p > 0.01) was determined between the above variant and variant 2 -Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed).All other variants obtained highly significant (p < 0.01) larger number of infected and decayed plants due to the attack of the parasitic fungi P. debarianum.Average number of infected plants in the three trial years in treated variants was 11.70% or 56.16% lower than in the three-year average in the control variants which was 26.69%.The difference in decayed plants was 70.97%.

Table 4 .
Root yield (t/ha) and sugar content (%) on two soil types during three trial years Table5) was recorded on Mollic Gleysols in the variant 7 -seed inoculated with P. fluorescens No 8569 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. subtilis No 2109 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. megaterium No 2894 (0.4 x 10 10 bacteria/ha).Average sugar content in molasses in treated variants was 2.60% or 14.75% lower than the average sugar content in molasses in the control variants (3.05%).

Table 5 .
Whipps (2001)l.(1987)andsugar yield (t/ha) on two soil types during three trial years By drawing comparison among the variants with the strains of the beneficial bacteria applied one at a time, the best results in this parameter as well were recorded with the bacteria P. fluorescens.The variants comprising the bacteria B. subtilis and B. megaterium, being applied one at a time, or together, obtained highly significant (p<0.01)lowervalues in all tested parameters.This is in agreement with the results ofLifshitz et al. (1987)andWhipps (2001)who stated that P. fluorescens as plant growth -promoting bacteria increased the solubility of phosphorus in inorganic forms around the active root zone.By mobilizing soil phosphorus plant vigour improves and yield increases by 15-20% (great root availability reduce negative nitrogen effect on sugar beet achieving balanced plant nutrition and reduced production of alpha -amino nitrogen, potassium and sodium i.e. decrease in the amount of sugar which cannot be isolated in the production process but turns into molasses).The lowest average sugar content in molasses in the three year trial was recorded on Eutric Cambisols with the variant 8 -seed inoculated with P. fluorescens No 8569 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. subtilis No 2109 (0.4 x 10 10 bacteria/ha) + seed inoculated with B. megaterium No 2894 (0.4 x 10 10 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed).Between this variant and variant 2 -Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed) no statistical significance (p>0.05) was determined.All other variants obtained highly significant (p<0.01)higher values of sugar in molasses.Average sugar content in molasses in treated variants was 3.39% or 14.61% lower than average sugar content in molasses in the control variants (3.97%).