Screening for Pseudomonas and Bacillus antagonistic rhizobacteria strains for the biocontrol of Fusarium wilt of chickpea

Received : 08.12.2015 Accepted : 20.01.2016 The aim of this work is to study the ability of several isolates belonging to Rhizobacteria (Pseudomonas and Bacillus) collected from several chickpea growing areas in Algeria, to control the mycelium growth of Fusarium oxysporum f. sp. ciceris. Interesting isolates were characterized for their morphological characteristics, physiological and biochemical activities as potential bio-control agent. Fungal inhibition tests were performed using plate assay and each isolate were tested for the production of protease, cyanide hydrogen, indole acetic acid, antifungal volatile and extracellular compound. According to API 50 CH, we are able to identify six Bacillus species (B. subtilis, B. circulans, B. lentus, B. aneurinilyticus, B. firmus, B. licheniformis; and with API 20NE test we have identified three Pseudomonas species (P. aeruginosa, P. luteola, P. fluorescens). The ability of bacterial isolates was varied in production of Protease, Gelatinase, Amylase, Cellulase, Acid Indole acetic, Lipase, Catalase and Cyanid Hydrogen. This is traduced in different rate of inhibition growth due to various extracellular compounds, where B61 (Bacillus aneurinilyticus) and P39 (Pseudomonas luteola) and P70 (Pseudomonas fluorescens) were the most efficient with 77 and 55.5% respectively, while B39 (Bacillus firmus) and P41 (Pseudomonas luteola) were the most efficient by volatile compounds with 70.5 and 77.5% respectively. Our results indicate that these bacteria isolates can be used in the biocontrol of Fusarium oxysporum f. sp. ciceris.


Introduction
Chickpea (Cicer arietinum L.) is an important pulse crop grown and consumed all over the world, especially in the Afro-Asian countries (Jukanti et al., 2012).Fungal plant pathogens are among the most important factors that cause serious losses to agricultural products annually (Ekundayo et al., 2011).Chickpea production is severely limited by Fusarium wilt which is caused by F. oxysporum Schlechtend.Fr. f. sp.ciceris (Padwick) Matuo and K. Sato.(Jalali and Chand, 1992).Fusarium wilt is a serious disease threat, especially in low rainfall areas, where weather conditions are favourable for disease development.From 33 countries of the world has been reported (Nene et al., 1996) causing 10-15% yield losses annually (Singh and Dahiya, 1973) depending upon the environmental conditions.The disease is more prevalent in the Indian subcontinent, United States, Tunisia, Algeria, Turkey, Ethiopia, Spain, and Mexico (Halila and Strange, 1996;Labdi, 1990;Nene et al., 1989;Westerlund et al., 1974).In severe cases, yield losses riches 100% under favourable conditions in chickpea (Landa et al., 2004).Other species and formae specialis of Fusarium also cause wilt in chickpea and produce mycotoxins (Gopalakrishnan et al., 2005), which markedly reduce the potential of crop rotation as a disease management strategy.Fusarium oxysporum f. sp.ciceris (FOC) may survive in soil and on crop residues as chlamydospores for up to six years in the absence of susceptible host, and spread by means of both soil and infected seed (Haware et al., 1978).
It is difficult to manage the disease either through crop rotation or application of chemicals because of soil nature persistence and its capacity to survive for long time even in the absence of host (Haware et al., 1996).Efficacy of wilt management was improved when bio-control agents were combined with cultural practices such as sowing date (Landa et al., 2004).Biological control provides an alternative to the use of synthetic pesticides with the advantages of greater public acceptance and reduced environmental impact (Reino et al., 2008).However, management of fungal diseases using antagonistic microorganisms, known as biological control, has been the focus of intense research worldwide (Killani et al., 2011).The use of bacteria as biocontrol agents of soil borne plant pathogens, as an alternative or complementary strategy to physical and chemical disease management, has been investigated for over 70 years (Weller, 1988).The lack of consistency in performance of beneficial bacteria such as Pseudomonas spp. or Bacillus spp.under field conditions has limited their use in commercial agriculture (Raaijmakers et al., 2002).Much of that inconsistency has been attributed to variability in physical and chemical properties within niches occupied by biocontrol agents that affect both colonization and expression of biocontrol mechanisms such as antibiosis, parasitism, competition and induced resistance.
Various mechanisms are involved in the biological control of fungal pathogens by Plant Growth Promoting Rhizobacteria (PGPR).These mechanisms include the production of secondary metabolites such as antibiotics, siderophores, hydrolytic enzymes, volatile extracellular metabolites, hydrogen cyanide and competition for nutrients, promotion of plant growth and, finally, induced resistance within the plants (Moeinzadeh et al., 2010;Kloepper et al., 1992) Therefore, biological control offers potential for suppression of Fusarium wilt under field conditions, particularly when used in combination with cultivars with partial resistance to the disease and choice of sowing date.Species of Bacillus have also been known to produce compounds which promote plant growth directly or indirectly, hydrogen cyanide (HCN), siderophores, indole acetic acid (IAA), solubilize phosphorous and antifungal activity (Saharan and Nehra, 2011;Wahyudi et al., 2011;Godinho et al., 2010).The objectives of this research were: (1) to characterize and select Pseudomonas and Bacillus isolates from rhizospheric and rhizoplanic soils infested with chickpea wilt, and (2) to determine their antagonistic activity in vitro in dual cultures against Fusarium oxysporum ciceris.

Preliminary screening
One hundred and forty for bacterial isolates were tested for their ability to produce antifungal substances against Fusarium oxysporum f. sp.ciceris using a dual-culture in vitro assay on PDA plates.Twenty μl of each bacterial suspension (10 8 cfu/ml) was placed on the plate.After 48h incubation at 28°C, a single 6 mm diameter mycelial disc was placed at the extremity of plates.Then, plates were incubated at 27-29 °C in darkness and after 5 days the growth diameter of the pathogen (distance between the point of placement of fungal disk and actively growing edges of the fungus) was measured.The percentage of growth inhibition was calculated using the method described by Erdogan and Benlioglu (2010).This experiment was conducted twice.Bacteria with inhibitory potential were selected for further experiments.

Identification of bacterial antagonist
Initially, the selected isolates were identified based on gram positive, spore forming, and fluorescent pigment production, aerobic or anaerobic growth.To identify Pseudomonas species, oxidase, catalase, amylase, protease, cellulase, indole acetic acid, lipase and gelatinase, growth at 41°C, growth at 4°C tests were further performed.To identify Bacillus, motility, growth at 45˚C, anaerobic growth in glucose broth, were assessed (Shaad, 1988).The Analytical Profile Index (API), particularly API 20E and API 5OCHB were used as supplementary tests, for the identification and differentiation of Pseudomonas, Bacillus and related species, respectively (Logan and Berkeley, 1984).

Protease production
Bacterial isolates were tested for protease production by growing them on skim milk agar (SKM) (Chantawannakul et al., 2002).The ability to clear the skim milk suspension in the agar was taken as evidence for the secretion of protease.Non-bacteria inoculated plates were used as control.

Hydrogen cyanide production
Production of hydrogen cyanide was determined on nutrient agar medium+ 4.4g of glycine.100 μl of bacterial culture (48 h) were streaked on the surface of medium, and then sterilized filter papers were soaked in 2.0% Na2CO3 in 5.0% (w/v) picric acid and placed in the upper lid of the Petri dish.The Petri dishes were sealed with parafilm and incubated at 30 °C for 4 days.A change in the colour of the filter paper from yellow to reddish brown was accepted as an index for cyanogenic activity.Non -inoculated plates with bacteria was used as control (Alstrom, 1987).

Indole acetic acid production (IAA)
The production of IAA was determined as described by Bric et al. (1991).Bacterial strains were inoculated into nutrient broth (peptone, 5 g; yeast extract, 1.5 g; beef extract, 1.5 g; and NaCl, 5 g; each per liter) and incubated at 30 °C for 5 days.A 5 ml culture was removed from each tube and centrifuged at 10,000 rpm for 15 min.An aliquot of 2 ml supernatant was transferred to a fresh tube to which 100 μl of 10 mM orthophosphoric acid and 4 ml of reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35% HClO4) were added.The mixture was incubated at room temperature for 25 min, and the absorbance of pink colour developed was read at 530 nm using a spectrophotometer.

Production of volatile antibiotics
Firstly, 100 μl of bacterial suspension (1×10 7 cfu/ml) from each isolate were sprayed on the surface of a Petri plate containing nutrient agar medium and incubated at 27-30 °C for two days.In another Petri plate containing PDA medium, a 5 mm disk of a 7 days-old pure culture of Fusarium oxysporum f. sp.ciceris was placed at the centre.Then both half plates were placed face to face preventing any physical contact between the pathogen and the bacterial suspension.Plates were sealed with parafilm.In the control plates, bacterial suspension was replaced with sterile water.Plates were incubated at 27-29 °C for 5 days and the percentage of inhibition zone was calculated for each isolates (Fiddaman and Rossall, 1993).For each treatment, there were four replicates and the experiment was repeated twice.

Data analysis
The results obtained were statistically processed through the analysis of variance ANOVA and B Tukey test at *P<0.05 and **P<0.01 to evaluate the significance between treatments.Correlation matrices have also been developed to define the interactions between the various parameters studied using SPSS version 18.0 and Microsoft Excel software 2010.

Extracellular and volatiles compounds
In dual culture test, Bacillus strains showed to have more inhibition of the pathogen growth in the PDA medium than Pseudomonas isolates.Indeed, with extracellular compounds diffused in the PDA medium, Bacillus strains (Table 1) gave an average of inhibition equal to 59.63 ± 9.99% vastly superior to 47.16 ± 9.32% recorded with Pseudomonas strains (Table 2).While, with volatiles compounds, Pseudomonas appears to be more effective in inhibition of the pathogen mycelium development.Thus, we obtained 42.15 ± 22.77% of mycelial growth inhibition by Pseudomonas isolates (Table 2) compared to 37.78 ± 16.55% recorded by Bacillus isolates (Table 1).If we look to gap-type calculated (22,77% and 16,55%), we must note here the wide intraspecific variability in both genera studied, particularly with volatile compounds it is more pronounced.As regards the connection between the percentage inhibition of mycelial growth and the inhibition zone, the results obtained do not show a direct bond because the largest zone of inhibition was not observed with the strains giving the higher level of inhibition either with Bacillus or Pseudomonas strains.Indeed, it was the strain B45, P31 and P39 that have gave the high read inhibition zones with 12 and 18mm respectively (Table 1 and 2).We can conclude, the general inhibition is due to the combined effects of several extracellular and volatile compounds.

Interspecific inhibition efficiency
In the case of Bacillus genera, the most efficient strains in the inhibition of mycelial growth were B61 with 77% of efficiency in extracellular compounds and B39 with 70.5% of efficiency in volatiles compounds (Figure 2).In the case of Pseudomonas, P53 was the most efficient in extracellular compounds (71%) and P41 (77.5%) in volatiles compounds tests (Figure 3).Regarding the interspecific efficiency, strain identified as Bacillus firmus appears as the more efficient (70,5%) in the inhibition of mycelia growth of Fusarium oxysporum f. sp.ciceris, both in extracellular and volatiles compounds tests.But the failure to identify a single strain, these results should be confirmed by interspecific comparison tests.With regard to Pseudomonas, there is very little difference between the three species identified by studying the effect of extracellular compounds, while in the case of volatile compounds, Pseudomonas fluorescens isolates seem to be more efficient with an average of 51 % of efficiency.
Both Bacillus and Pseudomonas isolates has gave a significant effect at 1% level according Tukey's test in inhibiting mycelia growth by extracellular compounds.These results were traduced by the appearance of three homogeneous groups and the distinction of B53, P31, P39, P53 and P70 isolates.

Production of antifungal metabolites
In vitro test using 32 bacterial strains against Fusarium oxusporum fs ciceris showed that different strains exhibited different combinations of antimicrobial metabolites.If all Bacillus strains have produced protease (Figure 4), amylase, catalase and Indole acetic acid excepting B47 isolate, however, they showed a great variability in the production of gelatinase, cellulase and lipase as an enzyme products and HCN as a volatile compound (Table 1).With regard to Pseudomonas strains, except catalase and HCN which all strains gave a positive reaction, we note a high qualitative and quantitative variability between species and strains of Pseudomonas, in protease production (Figure 4), gelatinase amylase, lipase and IAA products (Table 2).In the case of Bacillus strains, ANOVA analysis gave is a positive correlation between mycelia growth inhibition and inhibition zone, as well as for enzymatic activity between amylase and gelatinase.While, in the case of Pseudomonas strains, it has registered several positive correlations among different biochemical activities, example: the amylase is positively correlated with gelatinase and lipase, whereas the production of IAA is correlated with the cellulase and lipase production.From the principal components analysis (PCA), it emerges an entirely consistent correlation between biochemical tests and identification of bacterial species by API 50 CH and API 20NE.

Bacterial antagonism towards Fusarium species and strains
The results obtained show a great variability in inhibiting mycelial growth of the three FOC strains tested.Indeed, the results of direct confrontation in dual cultivation give the isolate FOC1 of the pathogen as the most sensitive fungal strain to antagonism of Bacillus isolates with an average reduction of mycelial growth equal to 58.16%.In addition, the rest of the Bacillus isolates do not exhibit a great difference in the effectiveness of the antagonism because the standard deviation is equal to 10.26 between the four deviations calculated.In contrast, the FOC2 behaves very differently when it is confronted with the Bacillus isolates,

A B
giving an average of reducing in mycelial growth equal to 40.87%.We have obtained zero inhibition with three strains of Bacillus ie B48, B65 and B69.This has resulted in a standard deviation of about 23.26.The FOC3 and Fusarium solani isolate gave intermediate sensitivity resulted in average reduction of mycelial growth of about 43.81% and 49.47%, respectively (Figure 5, 6).
Figure 5. Bacillus strains antagonistic effect on the hyphal growth in dual culture of Fusarium oxysporum f. sp.ciceris strains (FOC1, FOC2 and FOC3) and Fusarium solani.Tukey's test provides a significant effect at 1% and 5% level in the inhibition of mycelia growth with Pseudomonas and Bacillus isolates, respectively; thus resulting in the emergence of three distinct groups for each bacterial genus.While the effect of volatiles compounds was not significant at 5% level, that's mean, there is little quit diversity between strains tested.

Discussion
Fusarium oxysporum f. sp.ciceris is economically significant disease agent on chickpea.Due to the soil-borne nature of the disease, chemical applications for controlling the disease are rarely successful.Inconsistencies in biocontrol under varying environmental conditions have been a common limitation of soil borne pathogens.The present research was conducted to evaluate the efficacy of Pseudomonas and Bacillus species and strains against Fusarium oxysporum f. sp.ciceris.The bacterial strains are in most cases correlated and more efficient in the inhibition of the mycelail growth of Fusarium oxysporum f. sp.ciceris isolates, if they have the same agro-ecological origin; this makes them very effective in the biocontrol.Our results corroborate with those of Weller (1988) which states that the use of rhizobacteria in the control of plant diseases is most effective when rhizobacteria were isolated from the same rhizosphere of host plant.In this study, all bacterial isolates were selected from rhizosphere of chickpeas fields.Some bacterial isolates showed high inhibition activity on pathogen, whereas others showed only mild or no activity at all.About 39% (57 of 144) of isolates showed antagonistic activity against Fusarium oxysporum f. sp.ciceris in in vitro tests and giving more than 30% of inhibition of mycelia growth.The rest of isolates tested exhibited no or weak antagonistic activity against the pathogen on PDA plates assay.Similar results have previously been reported (Erdogan and Benlioglu, 2010;Tjamos et al., 2004;Khot et al., 1996).
Reduction of fungal growth by certain PGPR and formation of inhibition zones were presumably due to the antifungal substances and/or cell wall degrading enzymes; released by the bacteria into the culture medium (Fatima et al., 2009).Also, Sarhan et al. (2001) and Montealegre et al. (2005) pointed that the cell free culture filtrate of B. subtilis inhibited the mycelial growth, radial growth, and spore germination and germtubes length of F. oxysporum f.sp.ciceris.Many strains of Bacillus strains have been found to be potential biocontrol agents against fungal pathogens.This antifungal action involves the production of antibiotics, especially within soil microsites (Fravel, 2005).
Cyanide is a toxic and dreaded chemical produced by many rhizobacteria.Some bacteria synthesis it, others excrete it and yet others metabolize it in other to avoid predation and competition (Zeller et al., 2007).All Bacillus species and strains efficiency of inhibition of mycelium growth is due to the extracellular products than volatiles compounds.It has been reported that Bacillus strains have been able to inhibit the growth of a variety of fungal pathogens because of their ability to produce a vast array of antibiotics such as Zwittermicin, Bacillomycin, Fengycin, Bacilysin and Difficidin (Athukorala et al., 2009;Chen et al., 2009).While with Pseudomonas species and strains, Pseudomonas aeruginosa and Pseudomonas luteola join Bacillus in their action mode, whereas Pseudomonas fluorescens inhibits the development of the mycelium much more with volatile compounds.These results are in agreement with the research previously carried out by Romanenko and Alimov (2000).Production of Hydrogen cyanide in Bacillus is about 50% in both rhizospheric soils and nodules compared to Pseudomonas that is over 80% (Ahmad et al., 2005).This result demonstrated that cyanide hydrogen was one of the most important volatile compounds of Pseudomonas spp.Plant growth was enhanced in vitro by most of the rhizospheric isolated that produced HCN (Wani et al., 2007).
The majority of selected Pseudomonas and Bacillus strains showed amylase and protease activity pronounced comparatively to other metabolites productions.The protease is known that has interference in wall degrading of fungal pathogen (Ahmadzadeh and Sharifi-Tehrani, 2009).The Pseudomonas isolates had shown a high level of production of indole acetic acid comparatively to Bacillus isolates.Xie et al. (1996) indicated that compared to other strains, Pseudomonas strains had higher levels of IAA production.According to Joseph et al. (2007), while working with chickpea, all Bacillus isolates produced IAA.Even though some microorganisms produce high concentration of auxin, that is, IAA and this helps to increase plant growth and yield in wheat crop, others producing low concentration of IAA also improve plant growth (Tsavkelova et al., 2007).
Regarding bacterial antagonism towards Fusarium species and strains, our results agree with Adebayo and Ekpo (2005), because B. subtilis inhibited fungal growth and also promoted the growth of tomato plant in screen house trial.B. subtilis has been shown to have a broad spectrum of antimicrobial activities over diverse fungal and bacteria pathogen (Grover et al., 2009).This may be as a result of production of antibiotic, competition with pathogen for nutrients and direct antagonism (Akhtar et al., 2010).Bacillus spp.are known to reduce wilting index in F. udum, increase plant growth and cause rapid colonization of tomato tissue in order to induce systemic resistance against F. oxysporum (Kloepper et al., 2004).
As regards the behavior of FOC isolates and Fusarium solani in direct confrontation with Pseudomonas isolates in dual culture, it appears that the F. solani was the most sensitive with 53.06% of mycelia growth reduction followed by FOC1 isolate.The FOC2 is confirmed as the isolate which has the most variable behavior when confronted with different Pseudomonas isolates.Indeed, we noted that three isolates (P37, P53 and P65) did not any impact on mycelia growth.Besides that, we found that Bacillus and Pseudomonas strains are more effective in inhibiting mycelia growth if they came from the same geographical area with Fusarium strains.In general, F.oxysporum f.sp ciceris was more sensitive to bacterial metabolites and antagonisms than F. solani evidenced by the inhibition percentages recorded (Mudawi and Idris, 2014).
In conclusion, this study shows that some species and strains of Bacillus and Pseudomonas are very beneficial and effective as agents of biocontrol in vitro tests.The most interesting bacterial strains are being evaluated in vivo tests in the presence of the host plant for the study of their antagonistic faculty.Research must continue to be able to produce industrially as microbial biocontrol agents enjoying the characteristic of being respectful of human health of the environment in general.

Figure 1 .
Figure 1.Inhibition of mycelium growth of Fusarium oxysporum f. sp.ciceris by bacterial isolates on PDA media by dualculture assay (A Pseudomonas; B Bacillus).

Figure 2 .
Figure 2. Inhibition of mycelium growth of Fusarium oxysporum f. sp.ciceris, by extracellular and volatiles compounds of Bacillus species.

Figure 3 .
Figure 3. Inhibition of mycelium growth of Fusarium oxysporum f. sp.ciceris, by extracellular and volatiles compounds of Pseudomonas species.

Figure 4 .
Figure 4. Enzyme activities: Protease production by bacterial isolates on PDA media (A Pseudomonas isolate; B: Bacillus isolate).

Table 1 .
Antimicrobial metabolites activities of Bacillus isolate against Fusarium oxysporum f. sp.ciceris