Eur J Plant Pathol DOI 10.1007/s10658-017-1192-y
Interaction of root colonizing biocontrol agents demonstrates the antagonistic effect against Fusarium oxysporum f. sp. lycopersici on tomato Stuti Patel & Meenu Saraf
Accepted: 22 February 2017 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2017
Abstract Plant growth promoting Bacillus subtilis MSS9 and Bacillus licheniformis MSS14 were isolated from the tomato rhizosphere. These isolates were capable of inhibiting the fungal pathogen, Fusarium oxysporum f. sp. lycopersici causing fusarium wilt in tomato, tested by dual culture method and by mycolytic enzyme production. The isolates have the capacity to form biofilm on the microtitre plate. Scanning electron microscopy revealed good colonization capacity of Bacillus licheniformis MSS14 on tomato plant root as compared to Bacillus subtilis MSS9, pot experiments were also analyzed to study the effects of both rhizobacterial cultures on pathogen development and plant growth. It was observed that MSS14 reduces the incidence of Fusarium oxysporum f. sp. lycopersici in tomato and there was significant increase in vegetative parameters like root length, shoot length, plant wet weight, dry weight and chlorophyll content after which indicates that the root colonization property of the culture MSS14 helps in enhancing the biocontrol capacity against pathogen than that of MSS9.
Keywords Biofilm formation . Root colonization . Fusarium oxysporum f. sp. lycopersici (FOL) . Scanning electron microscopy (SEM)
S. Patel : M. Saraf (*) Department of Microbiology, University School of Sciences, Gujarat University, Ahmedabad, Gujarat 380 009, India e-mail:
[email protected]
Introduction Rhizospheric bacteria can have profound impact on plant health. Rhizosphere colonization is important not only as the first step in the pathogenesis of soil borne microorganisms, but is also crucial in the application of microorganisms for beneficial purposes like biofertilization, phytostimulation, biocontrol and phytoremediation. Colonizing microorganisms can be detected attached to the root in the rhizosphere or as endophyte. Biocontrol mechanisms of rhizobacteria are usually based on competition of nutrients and space, secretion of bioactive factors that attack the pathogen e.g., antibiotics, exo-enzymes, HCN, siderophores etc. and Induced systemic resistance (ISR) in plants are some of the mechanism that indirectly benefit plant growth. Colonization of large parts of the root system will facilitate biocontrol since colonization can be expected to function as the delivery system of antifungal metabolites (Lugtenberg Ben et al. 2001). Fomae speciales of Fusariumlike F. oxysporum f.sp.radicis-lycopersici Jarvis & Shoemaker (Kouki et al. 2012), F. oxysporum f. sp. lycopersici W.C. Snyder and H.N.Hans (Singha et al. 2011) and F. solani f. sp. eumartii (C. Carpenter) W.C. Snyder and H.N.Hans (Roomberg and Davis 2007) were found to be associated with wilt disease in tomato plants The root diseases of tomato have a significant impact on tomato production and present a considerable challenge for tomato producers. The fungus results in severe loss of tomato quality because of its ability to colonize the roots as it persist in soil by producing a resistant spore structure (Selvakumar et al. 2013).
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Biofilms are microbial communities in which a population of differentiated cells is encased by a self-made extra-cellular matrix (O’Toole et al. 2000). It is now widely recognized that most microbes associate with abiotic and biotic surfaces in the form of biofilms in natural, clinical and industrial settings (Davey and O’Toole 2000). Rhizosphere bacteria, plant growth promoting rhizobacteria (PGPR), are frequently found to form micro-colonies or biofilm-like structures on plant roots (Morris and Monier 2003). Effective colonization of plant roots by PGPR plays an important role in growth promotion, irrespective of whether the mechanism of action includes production of metabolites, production of antibiotics against pathogens, nutrient uptake effects, or induced plant resistance (Bolwerk et al. 2003; Raaijmakers et al. 1995). It is now common knowledge that bacteria in natural environments persist by forming biofilms. For suppression of root pathogens, root colonization property of introduced bacteria is required. Kloepper and Schroth (1981) showed that fluorescent pseudomonads after their introduction in the rhizosphere change the microbial community by reducing the population of pathogens. However, all the introduced bacteria do not prove to be successful in infecting, as this occurs due to the inability of the bacterium to successfully metabolize the root exudates and form the biofilms on the roots and even compete with the other microflora present in the soil (Latour et al. 1996). Hence, it has become necessary to develop such an inoculum which can successfully establish itself in the rhizosphere and can compete with the pathogen for niche. The present study has been carried out to investigate the root colonizing potential and biocontrol properties of rhizosphere bacteria against F. oxysporum f. sp. lycopersici.
Materials and methods Isolation of rhizospheric bacteria Soil samples were taken from the rhizospheric soil of tomato plants near Ahmedabad region of Gujarat and isolation were carried out using different medium (Jha and Saraf 2011). A total of 35 isolates were obtained which were taken to study their antagonistic effect against F. oxysporum f. sp. lycopersici (FOL) procured from MTCC Chandigarh.
In vitro biocontrol activity Biocontrol activities of all 35 isolates were tested against F. oxysporum f. sp. lycopersici by dual culture method. Results were expressed as means of radii of fungal growth (mm) and % inhibition of fungal growth in the presence of bacterial isolates (Berg et al. 2005). Percent inhibition was calculated using the following formula: %Inhibition ¼ ðA1−A2Þ=A1 100; Where A1 A2
Area covered by the pathogen in control, Area covered by pathogen in dual culture
Mycolytic enzyme assay The activity of cellulase and chitinase was determined by measuring the release of reducing sugars using carboxymethylcellulose and colloidal chitin as substrate. The reaction was carried out as per Chaiharn et al. (2009). Microtitre plate assay for biofilm formation Isolates were grown overnight in Luria Bertani (LB) medium and were diluted 1:100 into fresh medium for biofilm assays. Hundred μLof cultures per well were transferred to 96- well microtitre plates and incubated at 37 °C for 12 h and 24 h. After the incubation period, cultures were removed, and microtitre well plates were gently washed three times with sterile distilled water to remove loosely attached bacteria, and then dried at 30 °C for 30 min. Samples were stained by addition of 0.1% crystal violet to each well and incubated for 20 min. The intensity of crystal violet staining was measured after addition of 125 μL of 30% acetic acid to each well. After a further incubation period of 20 min, OD at 550 nm was measured on a plate reader (O’Toole 2011). All cultures were tested in triplicates. Gnotobiotic experiment and scanning electron microscope studies Seeds of Solanum lycopersicum L. (Tomato) were surface sterilized by 1% HgCl2 and bacterized with MSS9 and MSS14. Bacterized seeds were then sown in presterile soil for one month in a growth chamber to study
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the root colonization potential of respective isolates. After 30 days, roots were collected and fixed in glutaraldehyde according to Smyth et al. (1990). The samples were dehydrated through a graded ethanol series and critical point- dried in CO2. Pressure was decreased very slowly to prevent tissue damage. Root samples were mounted on stubs and shadowed with gold before viewing under FEG SEM (Nova Nano SEM 450 with EDAX). All images were computer processed. Genomic identification and phylogenetic analysis Both the isolates MSS9 and MSS14 having potential of in vitro biocontrol attributes against FOL, Biofilm formation and root colonization property were identified on the basis of 16srRNA sequence comparisons. Genomic DNA isolation was performed (Sambrook et al. 1989) and the complete 1.6-kb 16S rDNA region was amplified using the universal primer 1FAGCGGCGGAC GGGTGAGTAATG and 1509RAAGGAGGGGATCC AGCCGCA. DNA sequencing was performed using an ABI Prism Sequence Detection System. The BLASTn search program (http://www.ncbi.nlm.nih.gov) was used to look for nucleotide sequence homology. The sequences obtained were aligned by Clustal W using MEGA 6.0 software (Tamura et al. 2007) and a
neighbor-joining (NJ) and maximum parsimony tree was generated using the software. Pot experiment Tomato seeds were surface sterilized by 1% HgCl2 and washed five times with sterile distilled water. Seeds were coated with 1% carboxy methyl cellulose as adhesive. Seeds were then treated with both Bacillus sp. MSS9 and MSS14 and dried overnight at room temperature. Ten bacterized seeds were sown in small earthen pot filled with sterile sandy loam soil watered regularly. After 7 days of germination (Jha and Saraf 2011) seed germination percentage were recorded. Thirty days old seedlings were transplanted in the earthen pots containing sterile sandy soil, after 7 days of transplantation pathogen was inoculated as per the treatment as shown below. For each treatment, three such pots were maintained. After 30 days of challenge inoculation observations were recorded for root length (cm plant−1), shoot length (cm plant−1), chlorophyll content, plant wet weight (g), dry weight (g) and Vigour index for each treatment was calculated according to the formula suggested by Abdul-Baki and Anderson (1973). The percentage of disease incidence was calculated as per the formula by Tank and Saraf (2008).
Vigour index ¼ ½Mean of root length ðcmÞ þ Mean of shoot length ðcmÞ percentage of seed germination %Disease incidence ¼ No:of infected plants=No:of total plants 100
Treatments to study biometric parameters of Tomato against F. oxysporum f. sp. lycopersici.
levels of 5%. The ANOVA indicated significances of treatment and effects.
T1 T2 T3
Results
T4
MSS9 + F. oxysporum f. sp. lycopersici MSS14 + F. oxysporum f. sp. lycopersici Untreated soil and untreated seeds (Negative control) F oxysporum f. sp. lycopersici (Positive control)
Statistical analysis To evaluate the efficiency of rhizobacteria in pot experiments under biotic stress conditions, a completely randomized block design was used. To identify significant treatment effects, analysis of variance (ANOVA) was carried out. Mean values were compared at significance
Biocontrol attributes A total of 35 isolates were obtained from the rhizospheric soil of tomato. Out of them, 11 isolates were showing antagonistic activity against F. oxysporum f. sp. lycopersici (FOL). Among those, two isolates showed maximum inhibition of fungal growth were MSS9 and MSS14. The percentage of inhibition observed by MSS9 and MSS14 was 83% and 84.5% respectively against F. oxysporum f. sp. lycopersici as compared to other
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isolates (Fig. 1). Mycolytic enzymes such as cellulase and chitinase activity of both the isolates were also measured quantitatively by spectrophotometric method. It was found out the cellulase activity shown by MSS9 and MSS14 were 0.6 and 0.91 U/mL and that of chitinase activity were 0.25 and 0.42 U/mL respectively (Fig. 2).
as B. licheniformis (Weigmann) Chester (Genbank accession number KM376154). The results of the BLAST search program revealed that isolate MSS9 had a sequence homology with B. subtilis (Fig. 5a) and isolate MSS14 had a sequence homology with B. licheniformis (Fig. 5b). Pot experiment
Biofilm formation on microtitre plate Crystal violet assay for biofilm formation on solid surfaces was performed on microtitre plates to measure the biofilm formation (Friedman and Kolter 2004) as shown in Fig. 3 It has been observed that MSS14 showed maximum biofilm formation on the titre surface as compared to MSS9 measured in the form of optical density after 12 h and 24 h of incubation. Scanning electron microscopy The ability of the selected isolates to form biofilms on the solid surface as compare to the untreated plant root (Fig.4a). It was found that isolate MSS14 was more efficient at forming extracellular matrices (Fig. 4b). The SEM results confirm the result on abiotic surface showing that the isolate MSS14 is an effective biofilm former as compared with MSS9 (Fig. 4c). Identification of the selected isolates Selected isolates having both biocontrol activity against FOL and colonizing property on plant roots were identified on the basis of 16srRNA sequencing. MSS9 was identified as B. subtilis (Ehrenberg) Cohn (Genbank accession number KM376153) and MSS14 was identified
Fig. 1 Isolates showing percentage inhibition of F.oxysporum f. sp. lycopersici by dual culture method
Treatment with FOL showed decreases in root length and shoot length of 52% and 46% respectively when compared with untreated soil. Plants grown in the presence of pathogen without treatment with PGPR showed an almost 63% reduction in root length and about 60% reduction in shoot length in comparison to tomato plants grown with MSS14 while with that of MSS9 there was 60% and 56% reduction in root and shoot length respectively. Plant wet weight and dry weight was also calculated and it has been found out that there was increase in wet weight and dry weight of plant with untreated soil (−ve control), and soil inoculated with MSS9 and MSS14 along with pathogen when compared with the plant treated with only pathogen inoculated soil (+ve control). Total chlorophyll content also was observed to be increased in MSS9 treatment followed by MSS14 compared to that of positive and negative control as shown in Table 1. Seed germination rate was recorded maximum in the plants treated with B. licheniformis MSS14 infested with F. oxysporum f. sp. lycopersici (FOL) as compared to the soil treated with B. subtilis MSS9, Both positive control and negative control were maintained as shown in Table 2. There was 83% disease incidence observed in the plant challenged with F. oxysporum f. sp. lycopersici, while the plant treated with pathogen and
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Enzyme Prduction (U/mL)
Fig. 2 Mycolytic enzyme production of B. subtilis (MSS9) and B. lichenifomis (MSS14)
1 0.8 0.6 0.4 0.2 0 MSS9
MSS14
Isolates Cellulase units/mL
rhizobacterial isolates showed reduction in disease incidence. Percent disease incidence in plants treated with isolate MSS14 was 36.87% in tomato plant as compared to plants treated with isolate MSS9 was 52.08% as shown in Table 2.
Discussion Present research work was systematically carried out to test the antagonistic attributes, biofilm formation of the isolates and their role in disease suppression in plants
Chitinase units/mL
against phytopathogen. Isolation of native strains was conducted from the rhizosphere of tomato plants from the region close to Ahmedabad, India onto different isolation media. They were further studied for their biocontrol potential. Eleven isolates showed antagonistic behavior against F. oxysporum f.sp. lycopersici. Out of them two isolate B. subtilis MSS9 and B. licheniformis MSS14 had maximum antagonistic activity as shown by dual culture method. Karimi et al. (2012) reported antagonistic effects of 6 isolates of Pseudomonas and 6 isolates of Bacillus genera isolated from rhizosphere of chickpea against F. oxysporum f. sp. ciceris evaluated as
2.5
Fig. 3 Quantitative estimation of biofilm formation on microtitre plates by B. subtilis (MSS9) and B. lichenifomis (MSS14)
O.D. at 550nm
2
1.5
1
0.5
0
12hrs
24hrs
Time MSS9
MSS14
Eur J Plant Pathol Fig. 4 Scanning electron micrographs of tomato roots colonized by rhizobacterial isolates (a) showing untreated plant root (b) Plant roots colonized with isolate MSS14 (c) Plant roots colonized with isolate MSS9
a
b
c
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a
|KJ883532.1|_Bacillus_subtilis_strain_M.CH.V |KJ957013.1|_Bacillus_subtilis_strain_SH2 63
|KU132387.1|_Bacillus_subtilis_strain_YS15 |KT462749.1|_Bacillus_sp._BAB-4895 MSS9 |KJ726769.1|_Bacillus_licheniformis_strain_TG116 |KJ174691.1|_Bacillus_subtilis_strain_JNT02 |KJ870192.1|_Bacillus_amyloliquefaciens_strain_B12 |emb|X80725.1|_E.coli_(ATCC_11775T)
0.02
70 |HM589859.1|_Bacillus_sp._CK1
b
60 64 64 52
|KF555630.1|_Bacillus_sp._MN10 MSS14 |HM753633.1|_Bacillus_licheniformis_strain_WSR-KSU301 |JN934393.1|_Bacillus_sonorensis_strain_PDRRB5 |KM378585.1|_Bacillus_licheniformis_strain_BCX-51 |KJ526829.1|_Bacillus_licheniformis_strain_HT-17 |emb|X80725.1|_E.coli_(ATCC_11775T)
0.02
Fig. 5 a Phylogenetic analysis of MSS9 based on 16S rRNA gene sequences available from NCBI data library constructed after multiple alignments of data by Clustal W. Distances and clustering with the NJ method were calculated by using MEGA 6.0. Bootstrap values based on 500 replications are listed as percentages at the branching points. b Phylogenetic analysis of MSS14 based on
16S rRNA gene sequences available from NCBI data library constructed after multiple alignments of data by ClustalW Distances and clustering with the NJ method were calculated by using MEGA 6.0. Bootstrap values based on 500 replications are listed as percentages at the branching points
potential biocontrol agents in vitro and in vivo. Similarly, Selvakumar et al. (2013) study the antagonistic activity of P. fluorescens and B. subtilis on plant fungal pathogens, belonging to species F. oxysporum in dual culture test in vitro condition which provided the basis for the study of biocontrol effect in a pot culture experiment. Isolates
having maximum antagonistic ability were further studied for other biocontrol property such as chitinase and cellulase production.For an effective suppression of plant disease root colonization is important to reach the site to compete with the pathogen (Markovich and Kononova 2003). Thus, the isolates were further checked for their
Table 1 Yield parameters of different treatment on tomato plant against F. oxysporum f. sp. lycopersici Treatments
Root length (cm)
Shoot length (cm)
Plant wet weight (g)
Plant dry weight (g)
Total Chlorophyll content (mg/g)
Control
9.8c
16bd
23ac
22d
0.84a
MSS9
cd
14.6
24.3
28
1.03b
a
25.2
24.8
32
26.5
1.09bd
MSS9+ FOL
11.8ab
20.2d
24c
22.5ad
0.90b
FOL
12.9 b
4.7
a
21.8
bc
8.7
cd
ac
16.3
c
a
b
MSS14 MSS14+ FOL
e
a
bd
24.3
b
a
26
9.7
c
8.4
0.86a 0.56ac
Results are expressed as means of three replicates. Different letters indicate significant differences among treatment results taken at the same time interval according to Duncan’s multiple range test at p ≤ 0.05
Eur J Plant Pathol Table 2 Seed germination rate, vigor index and percentage of F. oxysporum f. sp. lycopersici incidence in differently treated tomato plants Treatments
% Seed Germination
Vigor Index % Disease Incidence
MSS9
94
3656.6
-
MSS14
92
3781.2
-
MSS9+ FOL
80
2560
52.08
MSS14+ FOL 85
2949.5
36.87
Control
76
1900
-
FOL
56
795.2
83
Results are expressed as means of three replicates
biofilm forming attributes and colonization property using microtitre plate assay and SEM analysis respectively. Results on the microtitre plate showed that both isolates have an ability to form biofilm on abiotic surfaces but isolate MSS14 was more efficient in biofilm formation on the surface of plate than that of MSS9. SEM results confirms the results of biofilm formation on solid surfaces, this may lead to better root colonization by MSS14 in comparison to MSS9. Similar results were obtained by Haggag and Timmusk (2008) showing two strains of Paenibacillus polymyxa isolated from the peanut rhizosphere were able to form the biofilm and showed efficient root colonization that suppress the pathogens causing crown rot diseases. Simlarly, Compant et al. (2005) found that Burkholderia strains 21 and 23 showing lower antagonistic effect as well as inefficient in controlling the disease, but upon root colonization it has been reported to penetrate the vascular system and spread in the plant parts to release certain inhibitory substances, resulting in induced resistance response. Thus, biofilm formation on plant root reported to be a successful biocontrol agent that colonizes well on the host plant, which is the first step to suppress the diseases caused by soilborne pathogens. The 16S rRNA gene sequence was determined and compared with representative sequences of members of genus Bacillus sp. The strain MSS9 and MSS14 formed a separate branch in neighbor-joining and was grouped most closely to a cluster containing B. subtilis and B. licheniformis with 99% sequence similarity respectively. Pot experiment done using both the isolates with F. oxysporum f. sp. lycopersici showed better root length, shoot length, wet weight, dry weight, seed germination rate, reduction in disease incidence and chlorophyll content as compared to the untreated plant and the plants inoculated with only F. oxysporum f. sp.
lycopersici. Reduction in disease incidence in plants treated with MSS14 was better than MSS9 which may be due to the maximum root colonization property of MSS14 on tomato root has been observed. Similarly, Anjaiah et al. (2003) have recorded that Pseudomonas aeruginosa PNA1 (Schroeter) whose root colonization capacity was tenfold lower in susceptible cultivar than the moderately tolerant cultivar of pigeon pea and chick pea significantly reduces fusarium wilt on both the varieties against F. oxysporum f. sp. ciceris (Padwick) and F. udum Butler respectively. This may suggest that the MSS14 has the greater biocontrol ability against F. oxysporum f. sp. lycopersici reflects its higher population densities in the rhizosphere and maximum root colonization property as compared with the isolate MSS9.
Conclusion Root colonizing property by the rhizobacterial isolates is a significant factor for the interaction between plant pathogen and plant beneficial rhizobacteria. In this study, root colonization property of MSS14 showed good vegetative parameters and reduces the penetrance of the pathogen (FOL) in the tomato plant as compared to the isolate MSS9 having poor root colonization capacity. Hence, from the present study it has been revealed that root colonization by rhizobacteria plays an important role in the plant growth promotion and disease suppression against phytopathogens. Acknowledgements We are thankful to Department of Microbiology and Biotechnology, Gujarat University to encourage and help us with the required facility and work and British Petroleum International Pvt. Ltd. for financial support.
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