Biological Control of Root Pathogens by Plant- Growth Promoting Bacillus spp

At the present time, among the most important factors limiting production of different crops are soil-borne plant pathogens [1]. Which include the genera Pythium, Rhizoctonia, Fusarium, Verticillium, Phytophthora spp, Sclerotinia, Sclerotium, and Rosellinia [2]. By this reason, different methods have been used to control these pathogens [3]. Cultural practi‐ ces and chemical control using synthetic fungicides are the most used control methods [4], however, use of some of these synthetic products has caused various problems due to environmental pollution, with consequences such as toxicity to humans, as well as re‐ sistance of certain pathogens to these fungicides [5]. An alternative to reduce the effect of these plant pathogens is the use of antagonistic microorganisms such as: some species of the genusBacillus which is recognized as one of the most effective biological control agent because of their properties on pathogens growth inhibition [6-7]. Biological control has many advantages as an alternative in the integrated management of diseases such as little or no harmful side effects, rare cases of resistance, long-term control, completely or substantially eliminates the use of synthetic pesticides, cost / benefit ratio very favorable; prevents secondary diseases, not symptoms of poisoning and can be used as part of inte‐ grated disease management [8]. Generally, the mode of action of Bacillus is antibiosis by producing extracellular hydrolytic enzymes which decompose polysaccharides, nucleic acids, other way are: production of antibiotics such as bacitracin, polymyxin, and grami‐ cidin, [9-11], competition to occupy an ecological niche and metabolize root exudates on pathogens affecting their growth [12-13]. Also, activating plant resistance induction when


Introduction
At the present time, among the most important factors limiting production of different crops are soil-borne plant pathogens [1].Which include the genera Pythium, Rhizoctonia, Fusarium, Verticillium, Phytophthora spp, Sclerotinia, Sclerotium, and Rosellinia [2].By this reason, different methods have been used to control these pathogens [3].Cultural practices and chemical control using synthetic fungicides are the most used control methods [4], however, use of some of these synthetic products has caused various problems due to environmental pollution, with consequences such as toxicity to humans, as well as resistance of certain pathogens to these fungicides [5].An alternative to reduce the effect of these plant pathogens is the use of antagonistic microorganisms such as: some species of the genusBacillus which is recognized as one of the most effective biological control agent because of their properties on pathogens growth inhibition [6][7].Biological control has many advantages as an alternative in the integrated management of diseases such as little or no harmful side effects, rare cases of resistance, long-term control, completely or substantially eliminates the use of synthetic pesticides, cost / benefit ratio very favorable; prevents secondary diseases, not symptoms of poisoning and can be used as part of integrated disease management [8].Generally, the mode of action of Bacillus is antibiosis by producing extracellular hydrolytic enzymes which decompose polysaccharides, nucleic acids, other way are: production of antibiotics such as bacitracin, polymyxin, and gramicidin, [9][10][11], competition to occupy an ecological niche and metabolize root exudates on pathogens affecting their growth [12][13].Also, activating plant resistance induction when installed in the roots and leaves which induces plant to produce phytoalexins which give resistance against attack by fungi, bacteria and pathogenic nematodes [14], reducing in these ways disease incidence.

Overview of Bacillus
The genus Bacillus Cohn was established in 1872, initially with two prominent forming endospores species: Bacillus anthracis and B. subtilis, actually, this genus has suffered considerable taxonomic changes, until early 1900, taxonomists not only restricted the genus to endospore forming bacteria, having that the number of species assigned to this genus were 146 in the 5th edition of Bergey's Manual of Systematic Bacteriology.Subsequent comparison studies by Smith et al. and Gordon et al. over 1114 strains of aerobic bacteria forming endospores (PGPR) helped to reduce this number to 22 well-defined species same as reported in the 8th edition of Bergey's Manual of Systematic Bacteriology [15].Bacillales is the order to which Bacillaceae family belongs within the genus Bacillus.This genus is characterized by having a rod shape within the group of Gram positive [16][17], and is therefore classified as strict aerobes or facultative anaerobes [18] and integrated by 88 species [15].A feature associated with this genus is that it forms a type of cell called endospore as a response to adverse growth conditions which distorts the structure of the cell.This spore form is resistant to high temperatures and current chemical disinfectants [19].This genus is abundant in various ecological niches which include soil, water and air [18,20], it is also found as food contaminants.Generally, Bacillus species used in bio-control are mobile, with peritrichous flagella, but yet some species are of interest in human medicine (B.anthracis) which are characterized as being stationary [21].

Ecology and habits
Distribution and habitat of Bacillus are very diverse; some species have been isolated from soil micro-flora adjacent to plants rhizosphere, water, air and food as contaminants [18,20].Eco-physiological criteria commonly used to group different species such as vertebrate pathogens, insect pathogens, antibiotics producer, nitrogen-fixing, denitrifying, thermophilic, psychotropic halophilic, alkali and acidifies rows.For example B. thuringiensis is considered an insect pathogen and is used as a bio-pesticide, it has been isolated from soil, and abundantly found worldwide [22][23][24], in soil remains largely in the form of endospores [25], particles of dust in suspension [26], insect bodies sick or dead [27], also is found in stored products [28][29], food [30], marine sediments [32], and even as opportunistic human pathogen [33].Furthermore, Bacillus species are found abundantly in plant leaves [34][35][36][37][38].In conclusion, the Bacillus genus has a cosmopolitan distribution (

B. xerothermodurans Soil Unknown
Table 1.Sources and common habitats of aerobic endospore forming bacteria of Bacillus genus, [39].

Many species of
Most of these antibiotics are cyclic peptides composed entirely of amino acids, but some may contain other residues.However, a few antibiotic peptides are linear such as rhizocticins [64].Bacillus spp.produces also a range of other metabolites including chitinases and other cell wall-degrading enzymes [65][66][67][68], and volatiles compounds [68][69][70] which elicit plant resistance mechanisms [14,71].
The amount of antibiotics produced by bacilli class was approaching 167 [45]

Collection and isolation of Bacillus
Traditional tools for determining composition of the soil bacterial community and diversity are based largely on in vitro culture methods.Typically, solid organic medium is inoculated with dilutions of a suspension of soil, then incubated and the colonies obtained are purified further sub culturing into another medium [73].Heat treatment or pasteurization is the most used technique for selecting spores.These techniques are very powerful because they are selective to remove all non-spore forming microorganisms, and are very efficient for obtaining populations of bacteria from spores, recommended temperatures oscillate between 65 to 70 °C for 15 minutes [74][75].However, heat treatment has to be adapted to certain species because endospores of some strains of bacteria are more resistant to heat than others, while incubation time used can vary from 3 to 30 min [76].It is recommended to start heating at a relatively low temperature (70 or 75 ° C) and gradually increasing to achieve an optimum temperature [77].To isolate endospores, some authors have taken advantage of spore tolerance to diverse stress conditions, for example, Koransky et al. [78] concluded that treatment with ethanol (50%) for 1 h is an effective technique to selectively isolate spore-forming bacteria, as effective as heat treatment to 80 ° C for 15 minutes.Patel et al. [79] confirmed this finding by isolating Bacillus strains from food residues, both by heating at 65 ° C for 45 minutes and incubation with ethanol.Soil drying may also be used as a selective method to isolation by striking desiccation tolerance of spores, which can therefore survive for long periods of time under these conditions.Drying treatment is probably more gentle that heating or ethanol incubation.Eman et al. [80] reported that vegetative cells were killed by addition of chloroform (1% v / v) however; this technique has not been validated.An interesting selection process, which is different from classical heat treatment was developed by Travers et al. [81] for isolation of Bacillus thuringiensis, which makes use of ethyl (ethyl selection), B. thuringiensis is selectively inhibited by sodium acetate (0.25 M), while most unwanted sporeforming species allowed to germinate.Then all non-sporulating bacteria were eliminated by heat treatment at 80 ° C for 3 min.Subsequently, surviving spores are germinated on enriched agar medium.Even if some other species of Bacillus are also selected by this method, such as B. sphaericus and B. cereus, this technique is commonly used for studying the diversity worldwide of B. thuringiensis [22,77].A modification to the method promotes greater sporulation spore production by stimulating shock before applying stress.For example, some authors suggest suspending one gram of soil in 50 mL of sporulation medium after incubation at 37 ° C under stirring for 48 hours before killing vegetative cells by heat treatment [80], while others proposed in soil suspensions incubate the culture broth at different temperatures for 5 days to allow better maturation of spores [77].

Biochemical identification
Biochemical test were the traditional method for bacteria identification to specie level, after that, strains are located at the genus taxonomically, based on characteristics of colony growth in artificial medium, form cell unit, presence, number and orientation of locomotive units, Gram stain, spore form and specific environmental conditions of growth and finally the specific use of carbon sources (biochemical tests) gave its metabolic diversity (Table 2  and 3).
Table 2. Differential characteristics of Bacillus species with ellipsoidal spores (Group I), [39].+= 90 or more of strains positive catalase; -= 10 or more of strains negative catalese; d= substancial proportion of specie differ; ND= Not done; NG= no growth.

Molecular identification
Bacillus species with diverse physiological traits require development of biochemical tests for identification [82].But advances in chromatographic analysis using whole cell fatty acid methyl esters (FAME) profiles allows doing this technique sufficiently sensitive and reliable for grouping Bacillus to specie level [83][84].Identification has become even more sensitive, by analysis of ribosomal DNA regions (16S rDNA) sequencing [85][86][87], and sequence analysis of gyrase B (gyrB) which has proved immensely valuable information for phylogenetic analysis of bacteria [88][89][90].Using 16S rDNA sequence, have been identified  [96]; antagonistic to Botrytis cinerea in postharvest [97], in the biological control of Rhizoctonia solani, Fusarium spp.and Pythium spp.[98], as well as inductor of resistance mechanisms in plants [99].Bacillus licheniformis is reported as a fungicide against a variety of pathogens, both as a preventive and curative particularly leaf spots and blights, and a growth-promoting bacteria with production likely gibberellins [100].Bacillus subtilis is the most studied and has been reported as growth promoter and antagonistic to a variety of pathogens such as Phytophthora cactorum, Sclerotium cepivorum, Fusarium oxysporum, Rhizoctonia solani, Alternaria carthami, Phytophthora capsici, and Fusarium solani among others, in different cultures and evaluated in vitro, greenhouse and field level [101][102][103], so that Bacillus strains can be using as an alternative in biological control for management plant disease.

In vitro studies
Results of in vitro research using Bacillus spp. as biocontrol agent against various soil pathogens, have reported positive responses through observing a negative effect on pathogen growth (Figure 1), per example against Alternaria dauci and Rhizoctonia solani, foliage and soil pathogens, respectively.In the Table 4 and 5, are showed some effect on pathogen mycelia inhibition by action of Bacillus, up to 50% compared to treatment control.Furthermore, in the case of A. dauci, greater control was observed with biocontrol agents compared to chemical treatment.

Greenhouse studies
Results under greenhouse conditions, present good evidence of Bacillus as biocontrol source for pathogens involved in diseases of root and plant foliage, to cause a decrease in disease development in both incidence and severity.In table 6 is showed that application of Bacillus on carrot foliage allowed a control of A. dauci incidence up to 25%, which represents a control to 2 times more than the chemical treatment used for its control.Likewise Bacillus use has favored not only reduction of symptoms and therefore incidence, but also helps to promote plant growth, which is expressed in greater plant height, as shown in Figure 2, there is an increase in tomato plants height by effect of a microcapsule formulation of Bacillus applied in the management of disease caused by F. oxysporum and R. solani, in contrast to the use of synthetic chemicals [104].

Field experience
Most research has been conducted in laboratory or greenhouse, and virtually no field-level assessments have been reported.A study carried out using Bacillus spp.for control of diseases caused by soil fungi including: F. oxysporum, R. solani and P. capsici in pepper and tomato crops allowed control of diseases incidence as seen in Table 7, use of Bacillus at transplanting can reduce disease incidence in contrast to traditional treatments (fungicide application) as Folpat, Captan, Mancozeb much as 64% and 72% compared to untreated control with the most efficient strain, only 36 and 40 respectively with less efficient biological treatment.Furthermore, the suppressive effect was maintained over time or among harvest times, this indicates that Bacillus strains suppressed disease caused by soil fungi and maintained their remedial effect through harvest times as seen in Table 8 where disease incidence and severity was lower than that offered by the traditional treatment performed by the farmer.In the case of tomato same behavior was observed for disease development with respect to the presence of Bacillus, Table 9.The application in field of Bacillus sp on melon crops (Figure 3) for the management of disease caused by F. oxysporum, showed an effect in reducing disease incidence in 41% compared to the conventional chemical treatment (TA), and increases in yields of 26.5% higher than TA, however there were no significant differences in the brix degrees, consistency of fruit, but an increase in 12% in the number of fruits and 20% in the length guide, leaves and stem diameter was observed [105].

Effect on plant development and growth
The effects obtained by applying Bacillus as fungicide were positive for crop development, because Bacillus stimulated biomass production, increased number of flowers and fruiting mooring, as seen in Table 10, where Bacillus was applied, plants had an increase in height, flowering and fruiting compared to traditional crop management through use of synthetic agrochemicals.It is noteworthy that use only at the time of transplantation Bacillus and 20 days after the second application, which is manifested by loss of effect at 84 days, but yet still persists pathogen control, before such situation should be applied in successive moments to keep a Bacillus greater crop protection coverage.In a similar way, positive effects of Bacillus application were observed on crop yield, by prevent soil pathogen attack.In Table 11, is showed that application of Bacillus increased pepper yields in contrast to the traditional crop management by up to 74% cumulative assessment in three harvest times.Bacillus favors growth of different plant parts such as stems and leaf area (Tables 12 and 13, Figure 4).

Conclusions
Use and application of biological control agents, such as Bacillus spp.prevents negative effects of pathogen attack on crops, providing an attractive option for sustainable agriculture due to their stimulating effects on plant growth, biomass production and its potential to increase plant production.In this chapter are mentioned clear and efficient biocontrol of plant pathogenic fungi by Bacillus strains, as evidence of lower disease incidence and severity.For that reason, it is suggested that B. subtilis can be incorporated to integrated management disease, where these strain may be used as biocontrol agent as well as biofertilizer.

Figure 2 .
Figure 2. Average fruit yield of tomato plants cv.Floradade under greenhouse conditions subjected to different treatments with microcapsules containing strains of Bacillus subtilis, a chemical control (TQ) and a blank (TA).

Figure 3 .
Figure 3. Treatments effect with Bacillus subtilis in fields on melon crops with high incidence of Fusarium oxysporum.

Figure 4 .
Figure 4. Effect of Bacillussubtilis in the biomass production of pepper plants (root).Treatments Bacillus and treatments without Bacillus.

Table 4 .
In [46]o mycelia inhibition of Rhizoctonia solani with Bacillus spp.strains and Larrea tridentata extract.*Summateddozes of Bacillus strains were 1x10 6 cfu / ml, 1 without agrochemicals,[46].Values in the same column followed by different letters are significantly at p <0.05.

Table 5 .
In [44]o mycelia growth inhibition of Alternaria dauci by Bacillus spp.and chitosan-Larrea (Q-L) suspensions.*Strains of Bacillus subtilis.Values in the same column followed by different letters are significantly at p <0.05,[44].

Table 6 .
Product effect of Bacillus based biological products and chemicals on incidence and severity of Alternaria dauci on carrot plants under greenhouse conditions.

Table 7 .
[9]ect of Bacillus and commercial products on root rot incidence in Capsicum annum at different harvest times,[9].Variance analysis used transformed data by arcsine.B/TT= Bacillus vs traditional treatments; B/T = Bacillus vs Control[9].

Table 8 .
[9]ect of four strains of Bacillus and commercial products on severity of wilt and root rot by Fusarium spp., Rhizoctonia solani and Phytophthora capsici, on pepper (Capsicum annum) using scales to wilt and root rot,[9].

Table 9 .
Disease incidence and severity at harvest time of tomato plants cv.Florade subjected to different treatments with microcapsules containing strains of Bacillus subtilis.*Chemical control treatments; ** Absolute control treatment, Values with same letters area not statistically different (Tukey, p<0.01).[104].

Table 11 .
[9]ect of Bacillus strains on pepper (Capsicum annuum) cv.Caballero development at 99, 113 and 146 days after inoculation under field conditions.B / TT = Bacillus vs. traditional treatment,[9].Weed and Pest Control -Conventional and New Challenges

Table 12 .
Height and leaf area of tomato plants cv.Floradade subjected to different treatments with microencapsulated strains of Bacillus subtilis.*: Chemical control treatment; **Absolute control treatment, Values with same letters are not statistically different (Tukey, p≤0.01), [104].Biological Control of Root Pathogens by Plant-Growth Promoting Bacillus spp.http://dx.doi.org/10.5772/54229