Articles Service
Research article
Suppression of Fusarium Wilt Caused by Fusarium oxysporum f. sp. lactucae and Growth Promotion on Lettuce Using Bacterial Isolates
Department of Applied Plant Sciences, Interdisciplinary Program in Smart Agriculture, Kangwon National University, Chuncheon 24341, Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2021; 31(9): 1241-1255
Published September 28, 2021 https://doi.org/10.4014/jmb.2104.04026
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Naturally occurring bacteria have been suggested as a replacement for supplements and chemical pesticides to control plant diseases [1]. Various species of bacteria have been focused on because of their root-colonizing capacity as well as their catabolic adaptability and production of metabolites with antibacterial and antifungal efficacy [2]. Several species of soil and seed-borne plant pathogenic fungi, such as
Control of all these phytopathogens is hugely based on genetic resistance in the host plant, use of synthetic pesticides, and environmental factors as well as management of the plant pesticides [4].
However, the use of chemical pesticides leads to inequality in the microbial community and may create new strains of resistant pathogens against beneficial microorganisms [5]. Therefore, the beneficial effects of rhizobacteria towards various phytopathogens can be explored for sustainable crop production [6, 7].
Soil bacteria and fungi possess some vital processes such as nitrogen fixation, nutrient mineralization and mobilization, decomposition and denitrification. The motility of bacteria has great impact on their ability to thrive in soil and colonization in the beginning phases where movement and attachment to the root surface are crucial [8]. Hence, essential identification of bacteria mostly involves the determination of colony morphology, catalase and oxidase testing, Gram staining, Voges-Proskauer tests (IMViC), and utilization of sugars (IMViC) [9].
Characterization of bacterial isolates via biochemical assay using organic manure and solid waste degradation was carried out based on IMViC and catalase oxidase through testing [10].
Applying environment friendly biocontrol agents is a specific and natural way to control plant pathogens and increase crop production [11].Several studies have been reported regarding the suppression of plant pathogens under in vitro and in vivo conditions using
The main objectives this study were to (i) isolate the potential antagonistic rhizobacteria from various sources and (ii) suppress Fusarium wilt caused by
Materials and Methods
Soil Sample Collection and Isolation of Bacterial Isolates
In total, 25 soil samples were collected from Chuncheon (37°56'21.69'' N, 127°46'55.30'' E), Hongcheon (37°41'46.74'' N, 127°54'19.01'' E), and Hwacheon (38°06'37.99'' N, 127°41'14.02'' E) in Gangwon-do, Korea, during April to June 2014. Soil sample collection was performed following the method of Harley and Waid (1975)[18]. Soil samples were dug about 2-3 inches from the immediate vicinity of rice (
Fungi and Culture Conditions
The fungal pathogen (
Screening of Bacteria Antagonistic to Fusarium oxysporum f. sp. lactucae
All bacterial isolates were screened for antagonistic activity against tested fungal plant pathogens using dual plate culture technique [20] with slight modification. Briefly, 6 mm mycelial plugs of actively growing pathogens were placed on the center of plates (150 mm diameter) containing PDA medium, and then eight sterilized paper discs (6 mm diameter) were placed equidistantly about 1.5 cm from the edge of the same plate. Suspensions of 10 μl of each bacterial isolate were inoculated in the paper discs and incubated for 7 days at 28°C until the fungus in the control plate covered the edge of the plate. The plates without bacterial inoculation and containing fungal plugs only were considered as control. Scoring technique was applied to measure the mycelial growth inhibition of pathogen. Zero (0) indicates the bacterial isolates fully covered with hyphae of fungi, one (1) indicates the fungal hyphae at the edge of the bacterial colony and, two (2) indicates a clear fungal mycelial inhibition zone around the bacterial colony [21]. The isolates with a score of 2 were only considered as antagonists and were selected for further evaluation of antagonistic properties by different methods.
Determination of Percentage Inhibition of Mycelial Growth of Fungal Pathogens
Antagonistic efficacy of screened bacterial isolates was further evaluated by dual culture technique. Briefly, 6 mm mycelial plugs of actively growing pathogens were placed on the center of Petri dishes (90 mm) containing 25 ml PDA-TSA (1:1 v/v), and then three sterilized paper discs (6 mm) were placed equidistantly about 1.5 cm from the edge of the same plate. Each paper disc was inoculated with a 10 μl freshly grown bacterial suspension at the concentration of 108 CFU/ml. The plates without bacterial inoculation and containing fungal plug only were considered as control. The test was done in triplicate. The antagonistic effect was determined by measuring the size of the inhibition zones and the radial growth of fungal mycelium. The percent inhibition of growth over control was calculated using this formula:
Inhibition of Fungal Mycelium Proliferation by Broth Culture Assay
One milliliter of 48-hour-old bacterial culture and two discs of 6 mm of test fungi were inoculated in 50 ml of PDB and TSB (1:1 v/v) in a conical flask of 250 ml at 28°C on a rotary shaker at 150 rpm (replications were made thrice per isolate). Control represents the broth inoculated with fungus only. The differences in dry weights between the bacterium treated and the control cultures were recorded by passing dual cultures grown for 7 days through pre-weighed filter paper. The filter papers were dried for 24 h at 70°C and weighed. The experiment had a completely randomized design with three replications. The reduction in weight of the test fungi was calculated using this formula in percentage [22]:
Reduction in weight (%) = (W1-W2)/W1×100,
where, W1 represents the weight of the test fungus in control flasks and W2 with the bacterial antagonists.
Elucidation of Antagonistic Traits
Chitinase activity. Qualitative estimation of chitinase was carried out in chitin agar plates prepared and amended with 2% phenol red and isolates (10 μl) were inoculated into wells. The plates were incubated for 120 h at 25-29°C and the chitinase activity was indicated as clear halos around the inoculated holes. The magnitude of the activity was calculated by measuring the diameter of the zones. The test was repeated in triplicate for each isolate [23].
Protein hydrolysis. Skim milk agar plates (skim milk 100 g, peptone 5 g, agar 15 g, distilled water 1,000 ml) were prepared and inoculated with pure bacterial culture into wells. The inoculated plates were incubated at 28°C for 48 h, and the plates were observed for clear zones around the wells [24].
Pectinase and cellulase production. To determine pectinase and cellulose production, the media were prepared by adding 1% pectin and cellulose in basal medium (NaNO3 1 g, K2HPO4 1 g, KCl 1 g, MgSO4.7H2O 0.5 g, yeast extract 0.5 g, glucose 1 g, distilled water 1,000 ml, agar 15 g). Ten microliters of the bacterial cell suspension was inoculated into the wells made on the medium and incubated for 5 days at 28°C. Gram’s iodine solution (3%) was poured in the pectin and cellulose agar media and zones of clearance were observed against the dark blue background. A clear zone against the blue background indicated that the bacteria were positive for pectinase and cellulase production. The magnitude of the activity was calculated by measuring the diameter of the zones. The test was repeated in triplicate for each isolate [24].
Elucidation of Plant Growth-Promoting Traits
Hydrogen cyanide (HCN) production. Nutrient agar amended with 4.4 g/l glycine and bacteria was streaked (log phase) onto plates. A Whatman filter paper No. 1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed at the top of the plates which were then sealed with parafilm and incubated at 35-37°C for 4days. Development of orange to red color indicated HCN production [25].
Hydrolysis of starch. Starch agar plates (peptone 5 g, beef extract 3 g, soluble starch 0 g, agar 15 g, distilled water 1,000 ml) were prepared and inoculated with pure bacterial culture and incubated at 25-29°C for 48 h. After incubation, iodine (3%) was poured onto the plates. Formation of a blue-black color due to starch-iodine complex in the unutilized places of starch in the agar plates was indicated. Starch hydrolysis by the bacteria via production of amylase was indicated by a clear halo zone surrounding the bacterial colony on the starch agar medium. The test was repeated thrice for each culture and recorded [24].
Siderophore production. Siderophore production by bacterial isolates was detected by the universal method of Schwyn and Neilands (1987) [26] using chrome azurol S (CAS) media. CAS agar plates were prepared and inoculated with the 10 μl of exponentially growing test bacterial culture (0.5 OD at 620 nm) and incubated at 28°C for 7 days. Development of a yellow-orange halo around the colony was considered as positive for siderophore production. The test was repeated thrice for all the cultures and siderophore production efficiency (SPE) was calculated by the following formula:
Ammonia production. Bacterial isolates (50 μl of bacterial cell suspension) were grown in 30 ml peptone water broth (4%) for five days at 25-29°C. Two milliliters of culture supernatant was mixed with 1 ml Nessler’s reagent and a volume of this mixture was increased to 8.5 ml by addition of ammonia-free distilled water. Development of yellow-to-brown color indicated ammonia production, and the optical density was measured at 450 nm using a spectrophotometer. The concentration of ammonia was estimated using the standard curve of ammonium sulphate in the range of 0.1-1.0 μmole/ml.
Indole acetic acid production (IAA). IAA production was estimated using the method described by Bric
Ten percent exponentially grown bacterial strain culture was inoculated in 100 ml NB (or 50 μl cell suspension in 5 ml of the sterile peptone yeast extract broth (peptone 10 g, beef extract 3 g, NaCl 5 g), with varying concentrations of L-tryptophan ranging from 0 to 500 μg/ml in a 15-ml tube. The broth (2 ml) was collected at 24, 48, and 72 h and centrifuged at 2,700 g for 15 min followed by assay for quantitative measurement of IAA. Then, 1 ml of the cell-free supernatant was mixed vigorously with 1 ml Salkowsky’s reagent (1 ml of 0.5M FeCl3 in 50 ml of 35% HClO4-perchloric acid) along with two drops of orthophosphoric acid and the assay system was kept at room temperature (25-29°C) in dark for 20 min till pink color developed (in a 2-ml Eppendorf tube). Optical density was measured spectrophotometrically at 535 nm. The concentration of IAA in each sample was determined from the standard curve of IAA with the standards prepared in the range of 10-100 μg/ml of IAA [28].
Phosphate solubilization. Phosphate solubilization activity of the selected rhizobacterial isolates was detected by means of plate assay using Pikovskaya (PVK) agar, which results in a clear halo formation. A pure colony from a fresh culture of each isolate was inoculated at four equidistant points into each of the PVK-agar media using a sterile needle. The diameter of the clear halo zone was observed after 12 days of incubation at 28°C. Control plates were inoculated with sterile tryptic soy broth (TSB) only. The diameters of the colony and clearing zones around the colonies were measured. All the tests were replicated thrice. The solubilization index of the isolates was calculated with the formula given below:
Zinc solubilization. The selected antagonistic bacterial isolates were inoculated into modified PVK medium (ingredients g/l), (glucose 10.0 g, ammonium sulphate 1.0 g, potassium choloride 0.2 g, dipotassium hydrogen phosphate 0.2 g, magnesium suphate 0.1 g, yeast 0.2 g, distilled water 1,000 ml, pH 7.0) containing 0.1% insoluble zinc compounds ( ZnO, ZnCO3, and ZnS). The test organisms were inoculated on these media and incubated at 28°C for 7 days. The diameters of the clear zone around the colonies were measured. All the tests were replicated thrice. The solubilization index of the isolates was calculated with the formula given below:
Molecular identification and phylogenetic analysis. For the extraction of DNA, the bacterial cells were harvested from 10 ml overnight culture and pellets were lysed in 1 ml lysis buffer (25% sucrose, 20 mM EDTA, 50 mM Tris-HCl and 5 mg/ml-1 of lysozyme). Chromosomal DNA was extracted following the standard procedure [29]. Universal primers 27F and 1492R were used to amplify the 16 rRNA using PCR [30]. The PCR was carried out in a thermocycler using 35 amplification cycles at 94°C (45 sec), 55°C (60 sec), and 72°C (60 sec) with a final extension for 7 min at 72°C. Products obtained from the PCR were purified by using a Montage PCR Clean-Up Kit (Millipore, USA). Universal primers, 518F and 800R (Macrogen, Korea) were used to sequence the purified PCR products of approximately 1,400 bp through a big Dye Terminator Cycle Sequencing Kit v.3.1 (Applied BioSystems, USA). An Applied BioSystems model 3730XL automated DNA sequencing system (Applied BioSystems) at Macrogen Inc. Seoul, Korea was used to resolved the sequencing products. The sequences were compared using the NCBI (National Center for Biotechnology Information) BLAST (Basic Local Alignment Search Tool) program (http://www.ncbi.nlm.nih.gov/Blast) for identification of the isolates. All positions containing gaps and missing data were eliminated from the dataset. Best hit sequences were downloaded in FASTA format from the NCBI database to construct a phylogenetic tree using MEGA 6 software [31].
Disease suppression by rapid radicle assay. The bacterial isolates were cultured in TSB (tryptic soy broth) with shaking at 150 rpm at 28°C for 48 h for bacterial suspensions. Seeds of lettuce were surface sterilized with 5%sodium hypochlorite for 20 min, washed thrice with sterile distilled water and kept in Petri dishes with moist filter paper for 3-4 days at 25°C in darkness for germination. Uniformly germinated seeds were soaked in the bacterial suspensions (108 cells/ml-1) of isolates. The treated seeds of lettuce were placed on the margins of actively growing mycelia of
Greenhouse and Field Evaluations
Preparation of fungal pathogen inoculum and inoculation technique. The pure culture of targeted fungal pathogen,
Experimental design and treatments. The greenhouse and field experiments were set up in RCB (Randomized Complete Block) designs with five replications. Ten bacterial isolates along with positive and negative controls were evaluated for their growth promotion and disease suppression activities on lettuce under greenhouse conditions. For growth promotion experiments, the positive control was maintained by mixing the autoclaved soil with chemical fertilizer (18 N: 7 P: 9 K) of 1 kg/1,000 m2 and uninoculated soil was treated as negative control. Three controls, infected with pathogen, non-infected or healthy, and positive (sprayed with 0.2% solution of Mancozeb 75% WP twice at intervals of seven days) were used in the disease evaluation experiments. Two potential bacterial isolates were tested under field conditions for their growth promotion and disease suppression activities on lettuce.
Observations. Disease severity (S) for Fusarium wilt of lettuce was estimated (after 5 and 8 weeks of transplanting, respectively), as a wilting percent using the rating scale in which infected plants were classified according to numerical grades ranging from 0 to 4 as follows: 0 = healthy, 1 = ≤ 25% of plant leaflets are yellow and of vascular root bundles are dark brown, 2 = ≥ 26-50% of plant leaflets are yellow and of vascular root bundles are dark brown, 3 = ≥ 50-75% of plant leaflets are yellow and of vascular root bundles are dark brown and 4 = ≥ 76-100% of plant leaflets are yellow and of vascular root bundles are dark brown.
where, A, B, C, and D are the number of plants corresponding to the numerical grades 1, 2, 3, and 4, respectively, and 4T is the total number of plants (T) multiplied by the maximum discoloration grade 4, where T = A + B + C + D. Reduction percentage was calculated using the formula of Guo
Statistical analysis. One-way analysis of variance (ANOVA) was applied to analyze the data from in vitro and to determine the significance of treatment effects. The percent data and data set having value zero (0) were transferred into arcsine square root transformation before further statistical analysis to improve the homogeneity of the variance of the data. Where the F values were significant, post hoc comparisons of means were made using Duncan’s multiple range test (DMRT) at the 0.05 probability level. All statistical analyses were done using CROPSTAT version 7.2.3 [34].
Results
Culturable Bacteria in the Rhizosphere and Endosphere
Bacteria were obtained both from the rhizospheric portion of various crop plants as well as the root interior of oat plants. Ninety-five bacteria were isolated from rice, maize, barley, sesame and soybean rhizospheric soil; and 23 were recovered from oat root interiors (Table S1). The general isolation frequency was 3.37. The isolation frequency in rhizospheric soil samples of rice, sesame, soybean, maize and oat was 4.86, 4.33, 4.00, 4.00, and 2.50, respectively. The lowest isolation frequency (2.30) was recorded in oat root samples and the highest number of isolates was recorded from rice rhizospheric soil samples.
Screening of Antagonistic Bacteria
Out of the 118 isolates tested, 20 isolates showed antagonism against all the test pathogens. The number of isolates with a score of 2 was 14 against
In Vitro Inhibition of F. oxysporum f. sp. Lactucae by Bacterial Isolates
All the screened bacterial isolates possessed inhibition against the tested pathogenic fungi. The highest inhibition was recorded by EN21 and OR7 (Table 1 and Fig. 1). Moreover, all tested bacterial isolates showed biomass reduction in all tested fungi with varied rate of reduction. The mycelial biomass of all tested fungi was reduced to the highest degree in dual culture broths inoculated with bacterial isolate EN21 (Fig. 2).
-
Table 1 . Antagonistic efficacy of rhizobacterial isolates against
F. oxysporum f. sp.lactucae in dual culture assay.Isolates Fungal pathogens F. oxysporum f. sp.lactucae RR8 56.1d (5.0d-e) RR12 52.9e (4.3d-f) RR26 54.5de (4.7de) RR33 49.8f (3.3f) RR34 55.7d (5.3d) MR3 53.7de (5.0de) MR19 62.0c (3.7ef) OR7 66.3a (7.7c) OR19 63.1bc (5.7d) EN4 0.0g (0.0g) EN18 63.9a-c (8.7bc) EN20 65.5ab (8.3c) EN21 66.3a (10.0a) EN22 65.4ab (8.7bc) EN23 65.5ab (9.7ab) Control 0.0g (0.0g)
-
Fig. 1. Growth promotion of
Fusarium oxysporum f. sp.lactucae by selected rhizobacterial isolates in dual culture assay. (A) Control; (B) RR8; (C) RR12; (D) RR26; (E) RR33; (F) RR34; (G) MR3; (H) MR19; (I) OR7; (J) OR19; (K) EN4; (L) EN18; (M) EN20; (N) EN21; (O) EN22 and (P) EN23.
-
Fig. 2. Reduction in mycelial dry weight biomass of
Fusarium oxysporum f. sp.lactucae due to antagonism of rhizobacterial isolates. Values with different lowercase letters indicate significant differences atp ≤ 0.05. Error bars indicate the standard error of three replicates.
Elucidation of Antagonistic Traits
Fifteen bacterial isolates were tested for antagonistic traits viz., chitinase, protease, pectinase and cellulase production. Clearing of plates containing colloidal chitin as a sole carbon source by the bacterium around the colony was used to measure chitin hydrolysis. All isolates, except RR33 and EN4, showed strong chitinolytic activity (Table 2, Fig. S1). The isolates RR34 and EN4 were weak producers of chitinase. Starch hydrolysis was observed via zones of starch hydrolysis through the production of α-amylase. Clearing of starch agar plates containing starch as a sole source of carbon by the bacterium around the colony was used to measure starch hydrolysis. Out of 15 isolates, 13 isolates were producers of α-amylase. The isolates RR34 and EN4 demonstrated negative response to starch hydrolysis (Table 2, Fig. S2). Clearing of skim milk agar plates containing skim milk as a sole source of protein by the bacterium around the colony was used for qualitative detection of protease production. Out of 15 isolates, 14 isolates demonstrated positive response to protein hydrolysis. The isolate RR34 was found negative with regard to production of protease (Table 2, Fig. S3). Cellulose degradation was observed via zones of cellulose hydrolysis through the production of cellulase. Clearing of agar plates containing cellulose powder as a sole source of cellulose by the bacterium around the colony was used. Out of 15 isolates, 14 isolates demonstrated positive response to cellulose degradation. The isolate EN4 was found negative for the production of cellulase (Table 2, Fig. S4).
-
Table 2 . Antagonistic traits of selected antagonistic bacterial isolates.
Isolates Hydrolytic enzymes HCN production Siderophore production Chitinase Endozymes Protease Cellulase Pectinase α-amylase Catalase Oxidase RR8 ++ +++ ++ ++ - - +++ ++ ++ RR12 ++ +++ ++ ++ - + +++ ++ ++ RR26 +++ +++ +++ +++ - + +++ +++ +++ RR33 + +++ ++ ++ - + + + + RR34 - + - - - + +++ + + MR3 ++ +++ ++ ++ - +++ +++ ++ ++ MR19 ++ +++ ++ ++ - +++ +++ +++ +++ OR7 +++ +++ +++ +++ - +++ +++ +++ +++ OR19 +++ +++ +++ +++ - +++ +++ +++ +++ EN4 +++ - - - - +++ + ++ ++ EN18 +++ +++ +++ +++ - +++ +++ ++ ++ EN20 +++ +++ +++ +++ - +++ +++ ++ ++ EN21 +++ +++ +++ +++ - +++ +++ ++ ++ EN22 +++ +++ +++ +++ - +++ +++ + ++ EN23 +++ +++ +++ +++ - +++ +++ ++ ++ +++ = high, ++ = medium, + = low, - = negative producer.
Growth-Promoting Trait Elucidation of Plant
The formation of yellow-to-orange halos was indicative of siderophore production. All tested isolates, except RR8, were positive for siderophore production (Table 2, Fig. S5).
Bacterial isolates were grown in peptone water broth for detection of ammonia production. Tubes showing faint yellow indicated a small amount of ammonia production, and deep yellow to brownish color indicated a maximum amount of ammonia production. Out of 15 isolates, 12 isolates were positive for ammonia production (Table 3). The isolates RR8, RR12 and RR33 showed negative response to ammonia production. The production of ammonia by the isolates EN4 and EN21 was more evident than the other isolates (Table 3). Maximum ammonia produced by the isolates EN4 and EN21 was 5.7 and 5.6 μmole/ml, respectively (Fig. 3 and Table 3).
-
Table 3 . Growth promoting traits of selected antagonistic bacterial isolates.
Isolates IAA† NH3 production† Phosphate solubilizationα Zinc solubilizationα RR8 - - + + RR12 - - + + RR26 - ++ - - RR33 - - - + RR34 - + - ++ MR3 - ++ - +++ MR19 - ++ - + OR7 + ++ - - OR19 - ++ - ++ EN4 ++ +++ +++ +++ EN18 - ++ - - EN20 - ++ - - EN21 ++ +++ ++ - EN22 - ++ - - EN23 - ++ + +++ †+++ = strong, ++ = medium, + = weak and - = no production of IAA and NH3;
α+++ = strong, ++ = medium, + = weak and - = no solubilization of phosphate and zinc.
-
Fig. 3. Ammonia production by bacterial isolates. (A) Control; (B) RR8; (C) RR12; (D) RR26; (E) RR33; (F) MR3; (G) MR19; (H) OR7; (I) OR19; (J) EN4; (K) EN18; (L) EN20; (M) EN21; (O) EN23 and (P) RR34.
It was observed that out of 15 isolates, only three isolates OR7, EN4 and EN21 could produce IAA only when L-tryptophan was supplemented in the medium. IAA production by the isolates was determined after 72 h of incubation and maximum IAA produced was 8.6 μg/ml by the isolate EN4 when L-tryptophan concentration in the medium was maximum (500 μg/ml) (Fig. 4). In the growth medium with absence of L-tryptophan, IAA was not detected in any of the three isolates even after 72 h (Fig. 4 and Table 3). This shows that there is a direct correlation between IAA production and supplemented L-tryptophan in the medium.
-
Fig. 4. Indole acetic acid produced by selected bacterial isolates at 72 h of incubation in different concentrations of L-tryptophan supplemented in nitrogen free broth. Error bar denotes the standard error of three replicates.
The bacterial isolates that showed zones of clearance on PVK agar media were considered as phosphate solubilizers and the phosphate solubilization index of all 15 bacterial isolates is shown in Table 3. Out of 15 isolates, five isolates demonstrated phosphate solubilization activity. The isolates RR8, RR12 and EN23 showed low solubilization efficiency while the isolates EN21 and EN4 demonstrated medium and high solubilization efficiency, respectively. The solubilization index of EN4 and EN21 was 4.0 and 2.2, respectively. Quantitative estimation of solubilized phosphate by potent bacterial isolates, EN4 and EN21, was done by PVK broth method. The amount of solubilized phosphate by the isolates EN4 and EN21 was 376.0 and 173.3 mg/l, respectively (Table 3, Fig. S6).
For zinc solubilization, the results showed that only nine isolates out of 15 isolates could form clearing zones in plate assay. Zinc solubilization potential varied among bacterial isolates (Table 3). The isolate EN4 showed the highest potential of zinc solubilization both in zinc oxide and zinc carbonate-containing media. It produced a clear zone of 16.7 and 15.7 mm with solubilization index of 3.4 and 3.2 in plates containing zinc oxide and zinc carbonate, respectively (Table 3, Fig. S7). HCN production by the bacterial isolates was observed as a change in color of the filter paper from yellow to orange brown. None of the tested isolates was found positive to HCN production (Table 2, Fig. S8).
Molecular Identification of the Bacterial Isolates
The molecular analysis revealed that 15 isolates belonged to three groups, Firmicutes, Proteobacteria, and Actinobacteria (Fig. 5). Most of the antagonistic bacteria (13 isolates, 86.6% of total) belonged to the Firmicutes group. Phylogenetic analysis based on 16S rRNA gene sequences indicated that
-
Table 4 . Similarity scores between bacterial isolates and the highly matched type strain identified by neighbor-joining analysis.
Bacterial isolates Closest GenBank accession No. Closest GenBank taxa Similarity (%) RR8 (KU512890) AB073186 Paenibacillus peoriae 99.5 RR12 (KU512891) AB073186 Paenibacillus peoriae 99.0 RR26 (KU512892) AF235091 Arthrobacter sulfonivorans 98.6 RR33 (KU512893) AB073186 Paenibacillus peoriae 99.1 RR34 (KU512894) AB073186 Paenibacillus peoriae 99.1 MR3 (KU512895) AB271744 Bacillus subtilis 99.7 MR19 (KU512896) AB271744 Bacillus subtilis 100.0 OR7 (KU512897) GQ281299 Bacillus siamensis 99.5 OR19 (KU512898) AB325583 Bacillus amyloliquefaciens 99.8 EN4 (KU512899) AJ537603 Pseudomonas proteolytica 99.0 EN18 (KU512900) GQ281299 Bacillus siamensis 99.5 EN20 (KU5129101) GQ281299 Bacillus siamensis 99.4 EN21 (KU5129102) GQ281299 Bacillus siamensis 99.4 EN22 (KU5129103) GQ281299 Bacillus siamensis 99.4 EN23 (KU5129104) GQ281299 Bacillus siamensis 99.4
-
Fig. 5. Phylogenetic analysis of internal transcribed spacer regions (16S rRNA gene sequences) of rhizobacteria isolated from various places in Gangwon-do, Korea. MEGA 6 software was used to construct the phylogenetic tree. Boldface indicates the sequences obtained in this study. Numerical values (>50) on branches indicates the percentage of 1,000 bootstrap replicates that support the branch. The scale bar expressed the number of changes per site.
Suppression of F. oxysporum f. sp. lactuace and Growth Promotion on Lettuce under In Vitro and In Vivo Conditions
The growth of lettuce seedlings with and without bacterial inoculation, based on root and shoot length and dry weight of whole plant, after 14 days of treatments, is presented in Table 5. The seed inoculations with bacterial strains increased the mentioned growth parameters over negative control and the increment was significant (
-
Table 5 . Efficacy of bacterial isolates on lettuce seedling growth by test tube method in vitro.
Isolates Shoot length (cm) Root length (cm) Seedling weight (mg/seedling) Fresh Dry RR8 10.57d 11.60ef 775.77g 45.03d RR12 10.43d 10.80fg 583.03k 40.10d RR26 9.47e 9.80h 552.70l 32.02e RR33 7.40f 10.07gh 549.87l 30.69e MR3 9.43e 11.00f 608.35j 43.36d MR19 9.60e 10.90f 630.43i 43.03d OR7 10.63d 12.47c-e 761.90g 45.20d OR19 10.53d 12.43c-e 707.68h 43.03d EN4 12.53b 13.33b 1235.90b 74.50a EN18 10.67d 12.33c-e 873.53f 55.13c EN20 10.97d 12.27c-e 1035.68e 54.23c EN21 12.20bc 13.03bc 1164.36c 65.35b EN22 11.67c 12.03de 882.70f 45.36d EN23 11.67c 12.70b-d 1128.36d 66.37b Positive Control 13.20a 15.07a 1321.83a 75.34a Negative Control 6.37g 8.30i 533.17m 30.70f Data are means of 10 replications.
Values with different alphabetic superscripts in the same column are significantly different at p ≤ 0.05 levels according to Duncan’s multiple range test.
-
Fig. 6. Efficacy of bacterial isolates on lettuce seedling growth by test tube method. (A) Negative control; (B) Positive control; (C) RR8; (D) RR12; (E) RR26; (F) RR33; (G) MR3; (H) MR19; (I) OR7; (J) OR19; (K) EN4; (L) EN18; (M) EN20; (N) EN21; (O) EN22, and (P) EN23.
-
Fig. 7. Disease occurrence caused by
Fusarium oxysporum f. sp.lactucae on radicles of lettuce seeds (cv.Jukchima ) treated with bacterial strains. Germinated lettuce seeds treated with distilled water (control) or bacterial suspensions for 2 h were placed on to the margin of actively growing mycelia ofFusarium oxysporum f. sp.lactucae on water agar containing 0.02% glucose for 7 days. Lowercase letters expressed the significant differences atp ≤ 0.05. The experiment was conducted with four replications of 5 seeds each. Square root transformed data were used for data analysis.
The results of the greenhouse experiment revealed that inoculation with bacterial isolates significantly promoted the growth of lettuce plants over negative control. However, the rate of enhancement varied with bacterial strains. Of tested isolates, isolate EN4 extensively increased all the growth attributes by recording 44.80 cm plant height, 1428.67 cm2 leaf area per plant, 38.40 chlorophyll content SPAD value, 1.80 g of root dry weight per plant, 6.35 g of shoot dry weight per plant and 20.50 cm root length (Table 6 and Fig. 8). The results were significantly higher than negative control and most of the bacterial isolates. The results revealed that the effects of isolates EN4 and EN21 were comparable to chemical fertilizer though all the crop attributes were significantly higher in plants treated with chemical fertilizer. Moreover, EN21 showed highest suppression (66.11%) of tested pathogen under greenhouse conditions (Table 7). The results also showed that plants inoculated with any of the tested bacterial isolates significantly reduced wilting percentage (Fig. 7). The highest disease severity reduction was observed with isolate EN21 and then by EN23. The reductions in disease severity by these two isolates were 66.11 and 60.68%, respectively (Table 7). The lowest reductions were produced by isolates EN4 and RR8 (26.21 and 32.06%, respectively). The isolate EN21 caused a 140.5% increment in dry shoot weight over infected control by reducing wilting (Table 7).
-
Table 6 . Effects of bacterial isolates on growth parameters of lettuce in soil treatments under greenhouse conditions.
Isolates Plant height (cm) Leaf area (cm2/plant) Chlorophyll content (SPAD value) Fresh weight (g/plant) Dry weight (g/plant) Root length (cm) Root Shoot Root Shoot RR8 34.17fg 1235.67h 31.03gh 8.47f 51.98g 0.85g 4.12ef 15.53g MR19 34.13fg 1230.33h 30.77h 8.44f 51.90g 0.82g 3.71f 15.53g OR7 35.57e 1330.00g 32.80ef 12.71e 53.48g 1.14e 4.69d 16.33f OR19 35.03ef 1327.00g 31.87fg 12.39e 53.22g 0.96f 4.58de 16.13fg EN4 44.80b 1428.67b 38.40b 18.73b 92.29b 1.80b 6.35a 20.50b EN18 35.47e 1346.33f 34.83d 13.52d 71.02d 1.29d 4.97cd 17.33de EN20 33.50g 1320.67g 33.47e 13.45d 62.01f 1.28d 4.71d 16.63ef EN21 42.67c 1415.67c 36.93c 18.51b 75.94c 1.72b 5.56b 18.50c EN22 35.87e 1362.67e 35.57d 14.64c 67.79e 1.50c 5.03cd 17.43d EN23 38.53d 1391.67d 35.13d 14.75c 71.32d 1.53c 5.37bc 18.30c Positive Control 51.63a 1530.00a 40.70a 20.70a 98.37a 2.87a 6.72a 21.47a Negative Control 31.17h 462.33i 28.50i 7.51g 41.43h 0.75h 2.85g 13.20h Data are means of five replications.
Values with different alphabetic superscripts in the same column are significantly different at p ≤ 0.05 levels according to Duncan’s multiple range test.
-
Table 7 . Effect of inoculation with rhizobacteria on development of Fusarium wilt and shoot dry weight on lettuce under greenhouse conditions.
Treatmentsa Disease severityb (%) Disease reduction (%) Shoot dry weight (g/plant) RR8 65.67bc 32.06 3.67g MR19 61.33c 36.56 3.67g OR7 45.33d 53.10 4.45e OR19 44.00de 54.47 4.47e EN4 71.33b 26.21 4.2f EN18 42.00de 56.54 4.85c EN20 44.00de 54.49 4.72d EN21 34.67fg 66.11 6.35a EN22 40.33def 58.27 4.93c EN23 38.00e-g 60.68 6.12b Chemical 32.67g 64.12 2.92h Non-infected Control - - 2.75i Infected Control 96.67a - 2.64j
-
Fig. 8. Shoot growth promotion on lettuce by bacterial isolates under greenhouse conditions. (A) Negative control; (B) Positive control; (C) RR8; (D) MR19; (E) OR7; (F) OR19; (G) EN4; (H) EN18; (I) EN20; (J) EN21; (K) EN22; and (L) EN23.
Inoculation of plants with
-
Table 8 . Effect of inoculation with rhizobacteria on development of Fusarium wilt and shoot length of lettuce under field conditions.
Treatmentsa Shoot length (cm) Disease severityb (%) Disease reduction (%) EN21 85.17b 45.9b 44.91 EN4+21 94.83a 35.7cd 57.15 Chemical 74.83c 30.5d 63.39 Non-infected Control 64.67d - - Infected Control 28.17e 83.33a - aLettuce plants (cv. Jukchima) were treated by drenching the soil around root zone with the broth culture of bacterial isolates two times at an interval of seven days. Control plants (not infected and infected control) were treated with tap water and plants were sprayed with 0.2% solution of Mancozeb 80WP two times at an interval of seven days.
bDisease severity was recorded at 8 weeks after planting.
Data are means of five replications. Values with different alphabetic superscripts in the same column are significantly different at
p ≤ 0.05 levels according to Duncan’s multiple range test.
-
Fig. 9. Effect of inoculation with rhizobacteria on development of Fusarium wilt and foliage yield of lettuce under field conditions.
Discussion
Soil microorganisms are regarded as an important and essential component of soil quality due to their crucial activities in many ecosystem processes [35, 36]. Rhizospheres have been frequently exploited as an excellent source of biocontrol agents, since they provide the frontline of defensive microorganisms for roots against the attack of soil-borne pathogens [37]. In this study, 20 antagonistic bacterial isolates out of 118 rhizobacterial isolates were screened with 13 fungal pathogens as targets. The antagonistic bacterial isolates exerted varied levels of antagonism against tested pathogens. Fluctuation in the spectrum of antifungal activity of bacteria is common [38]. In dual culture assays, isolates RR8, MR3, MR19, OR7, OR19, EN18, EN20, EN21, EN22, and EN23 showed maximum inhibition of radial growth of test pathogens. In this study, some bacterial isolates were found to be highly inhibitory of fungal growth whereas others showed only minor activity or no activity at all. The inhibition zone exhibited between the fungal pathogens and bacteria was expressed in the inhibition of fungal mycelium. Moreover, as the PDA medium used for the dual culture assay is rich in nutrients, competition might be excluded as the mode of action for these isolates [39]. The antifungal metabolites produced seems to vary among the bacterial isolates tested in this study. This suggests that the fungal mycelia might not only be inhibited by antibiosis but also by other antifungal metabolites such as siderophores, hydrogen ions and gaseous products including ethylene, hydrogen cyanide and ammonia [40]. In vitro broth-based dual cultures offer a better method for evaluation of antagonistic efficiency of the biocontrol agents as the liquid medium may provide a better environment to allow the antagonistic activities from all possible interacting sites. These results are in agreement with the findings of Ashwini and Srividya (2014) [41] who revealed that antagonistic bacteria,
This study revealed that some rhizobacterial isolates were capable of inhibiting a wide range of phytopathogens in controlled conditions. But, in most biocontrol investigations, a large number of antagonists are commonly isolated over a short period of time and screened in vitro for antagonistic activity and tests based on in vitro mycelial inhibition and root colonization do not always correlate with biocontrol efficacy under natural conditions [42]. However, little correlation exists between in vitro and in vivo antagonistic activity in general [43] and identification of promising field-effective bacteria, however, can be facilitated by greenhouse experiments [44]. The major bacterial genus identified in our studies was
In the present study,
Supplemental Materials
Acknowledgments
This study was conducted with the support of a research grant from Kangwon National University.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- Bano N, Musarrat J. 2003. Characterization of a new
Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent.Curr. Microbiol. 46 : 324-328. - Nelson EB, Maloney AP. 1992. Molecular approaches for understanding biological control mechanisms in bacteria: studies of interaction of
Enterobacter cloacae withPythium ultimum .Can. J. Plant Pathol. 14 : 106-14. - Armstrong GM, Armstrong JK. 1981. Formae speciales and races of
Fusarium , pp. 391-399.In: Nelson PE, Toussoun TA, Conk RJ (eds), . The Pennsylvania State University Press, University Park.Fusarium : diseases, biology and taxonomy - Ulloa M, Hanlin R. 1993. Plant disease control, pp. 448.
In: Strange R (ed),Plant Disease Control: Towards Environmentally Acceptable Methods , 1st ed. Chapman and Hall, New York. - Shanmugam V, Kanoujia N. 2011. Biological management of vascular wilt of tomato caused by
Fusarium oxysporum f. sp.lycopersici by plant growth-promoting rhizobacterial mixture.Biol. Control. 57 : 85-93. - Cavender ND, Atiyeh RM, Knee M. 2003. Vermicompost stimulates mycorrhizal colonization of roots of
Sorghum bicolor at the expense of plant growth.Pedobiologia (Jena) 47 : 85-89. - Jetiyanon K, Kloepper JW. 2002. Mixtures of plant growth-promoting rhizobacteria for induction of systemic resistance against multiple plant diseases.
Biol. Control 24 : 285-291. - Turnbull G a, Morgan JAW, Whipps JM, Saunders JR. 2001. The role of motility in the in vitro attachment of
Pseudomonas putida PaW8 to wheat roots.FEMS Microbiol. Ecol. 35 : 57-65. - Dubey RC, Maheshwari DK. 2005. Enhancement of collar rot in sunflower caused by
Sclerotinia rolfsii byPseuodomonas .Indian Phytopathol. 58 : 17-24. - Zaved HK, Rahman MM, Rahman MM, Rahman A, Arafat SMY, Rahman MS. 2008. Isolation and characterization of effective bacteria for solid waste degradation for organic manure.
KMITL J. Sci. Tech. 8 : 44-55. - Whipps JM. 1997. Ecological considerations involved in commercial development of biological control agents for soil-borne diseases, pp. 525-546.
In: van Elsas JD, Trevors, JT Wellington EMH (eds),Modern soil microbiology . Marcel Dekker, New York. - Arrebola E, Jacobs R, Korsten L, Iturin. 2010. A is the principal inhibitor in the biocontrol activity of
Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens.J. Appl. Microbiol. 108 : 386-395. - An Y, Kang S, Kim KD, Hwang BK, Jeun Y. 2010. Enhanced defense responses of tomato plants against late blight pathogen
Phytophthora infestans by pre-inoculation with rhizobacteria.Crop Prot. 29 : 1406-1412. - Júnior VL, Maffia LA, Romeiro RS, Mizubuti ESG. 2006. Biocontrol of tomato late blight with the combination of epiphytic antagonists and rhizobacteria.
Biol. Control. 38 : 331-340. - Akutsu K, Hirata A, Yamamoto M, Hirayae K, Okuyama S, Hibi T. 1993. Growth inhibition of
Botrytis spp. bySerratia marcescens B2 isolated from tomato phylloplane.Ann. Phytopathol. Soc. Jpn. 59 : 18-25. - Sutton JC, Peng G. 1993. Biocontrol of
Botrytis cinerea in strawberry leaves.Phytopathology 83 : 615-621. - Paul B. 1999. Suppression of
Botrytis cinerea causing the grey mould disease of grape-vine by an aggressive mycoparasite,Pythium radiosum .FEMS Microbiol. Lett. 176 : 25-30. - Harley JL, Waid JS. 1975. A method of studying active mycelia on living roots and other surfaces in the soils.
Trans. Br. Mycol. Soc. 38 : 104-118. - Bharathi R, Vivekananthan R, Harish S, Ramanathan A, Samiyappan R. 2004.
Rhizobacteria based bio-formulations for the management of fruit rot infection in chillies.Crop Prot. 23 : 835-843. - Sheng JX, Kim BS. 2014. Biocontrol of fusarium crown and root rot and promotion of growth of tomato by
Paenibacillus strains isolated from soil.Mycobiology 42 : 158-166. - Perneel M, Heyrman J, Adiobo A, De Maeyer K, Raaijmakers JM, De Vos P, Höfte M. 2007. Characterization of CMR5c and CMR12a, novel fluorescent
Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity.J. Appl. Microbiol. 103 : 1007-1020. - Trivedi P, Pandey A. 2007. Biological hardening of micropropagated
Picrorhiza kurrooa Royel ex Benth., an endangered species of medical importance.World J. Microbiol. Biotechnol. 23 : 877-878. - Roberts WK, Selitrennikoff CP. 1988. Plant and bacterial chitinases differ in antifungal activity.
J. Gen. Microbiol. 134 : 169-176. - Cappuccino JC, Sherman N. 2006. Microbiology: a laboratory manual, 6th ed. Pearson Education, Inc, San Francisco.
- Lorck H. 1948. Production of hydrocyanic acid by bacteria.
Physiol. Plant. 1 : 142-146. - Schwyn B, Neilands JB. 1987. Universal chemical assay for the detection and determination of siderophores.
Anal. Biochem. 160 : 47-56. - Bric JM, Bostock RM, Silverstone SE. 1991. Rapid
in situ assay for indole acetic acid production by bacteria immobilized on a nitrocellulose membrane.Appl. Environ. Microbiol. 57 : 535-538. - Goswami D, Vaghela H, Parmar S, Dhandhukia P, Thakker JN. 2013. Plant growth promoting potentials of
Pseudomonas spp. strain OG isolated from marine water.J. Plant Interact. 8 : 281-290. - Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 1991. 16S ribosomal DNA amplification for phylogenetic study.
J. Bacteriol. 173 : 697-703. - Reysenbach AL, Giver LJ, Wickham GS, Pace NR. 1992. Differential amplification of rRNA genes by polymerase chain reaction.
Appl. Environ. Microbiol. 58 : 3417-3418. - Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0.
Mol. Biol. Evol. 30 : 2725-27299. - Oh BJ, Kim KD, Kim YS. 1999. Effect of cuticular wax layers of green and red pepper fruits on infection by
Colletotrichum gloeosporioides .J. Phytopathol. 147 : 547-552. - Guo JH, Qi HY, Guo YH, Ge HL, Gong LY, Zhang LX. 2004. Biocontrol of tomato wilt by plant growth-promoting rhizobacteria.
Biol. Control 29 : 66-72. - Jeffries P, Gianinazzi S, Perotto S, Turnau K, Barea JM, IRRI. CROPSTAT for Windows, version 7.2.3. 2007; Metro Manila, Philippines. 2003. The contribution of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and soil fertility.
Biol. Fertil Soils 37 : 1-16. - Atkinson A, Watson CA. 2000. The beneficial rhizosphere: a dynamic entity.
Appl. Soil Ecol. 48 : 99-104. - Garbeva P, van Veen JA, van Elsas JD. 2004. Microbial diversity in soil: selection of microbial populations by plant and soil type and implications for disease suppressiveness.
Ann. Rev. Phytopathol. 42 : 243-270. - Paulitz TC, Zhou T, Rankin L. 1992. Selection of rhizosphere bacteria for biological control of
Pythium aphanidermatum on hydroponically grown cucumber.Biol. Control 2 : 226-237. - Bakthavatchalu V, Dey S, Xu Y, Noel T, Jungsuwadee P, Holley AK,
et al . 2011. Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Polγ against UV-induced inactivation.Oncogene 31 : 2129-2139. - Landa BB, Hervas A, Bethiol W, Jimenez-Diaz DR. 1997. Antagonistic activity of bacteria from the chickpea rhizosphere against
Fusarium oxysporum f. sp.ciceris .Phytoparasitica 25 : 305-318. - Saravanan T, Muthusami M, Marimuthu T. 2004. Effect of
Pseudomonas fluorescens on fusarium wilt pathogen in banana rhizosphere.J. Biol. Sci. 4 : 192-198. - Ashwini N, Srividya S. 2014. Potentiality of
Bacillus subtilis as biocontrol agent for management of anthracnose disease of chilli caused byColletotrichum gloeosporioides OGC1.3Biotech. 4 : 127-136. - Williams GE, Asher MJC. 1996. Selection of rhizobacteria for the control of
Pythium ultimum andAphanomyces cochlioides on sugarbeet seedlings.Crop Prot. 15 : 479-486. - Baker R. 1968. Mechanisms of biological control of soil-borne pathogens.
Annu. Rev. Phytopathol. 6 : 263-294. - Xu GW, Gross DC. 1986. Selection of fluorescent pseudomonads antagonistic to
Erwinia carotovora and suppressive of potato seed piece decay.Phytopathology 76 : 414-422. - Majeed A, Abbasi MK, Hameed S, Imran A, Rahim N. 2015. Isolation and characterization of plant growth-promoting rhizobacteria from wheat rhizosphere and their effect on plant growth promotion.
Front. Microbiol. 6 : 198. - Voisard C, Keel C, Haas D, Dèfago G. 1989. Cyanide production by
Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions.EMBO J. 8 : 351-358. - Bashan Y, De-Bashan LE. 2010. How the plant growth-promoting bacterium
Azospirillum promotes plant growth-a critical assessment.Adv. Agron. 108 : 77-136. - Muthamilan M, Jeyyarajan R. 1996. Integrated management of
Sclerotium root rot of groundnut involving ofTrichoderma harzianum ,Rhizobium and Carbendazim.Indian J. Mycol. Plant Pathol. 26 : 204-209. - van Peer R, Niemann GJ, Schippers B. 1991. Induced resistance and phytoalexin accumulation in biological control of fusarium wilt of carnation by
Pseudomonas sp. Strain WCS417r.PDF.Phytopathology 81 : 728-734. - Morrissey JP, Walsh UF, O'Donnell A, Moënne-Loccoz Y, O'Gara F. 2002. Exploitation of genetically modified inoculants for industrial ecology applications.
Antonie Van Leeuwenhoek 81 : 599-606.
Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2021; 31(9): 1241-1255
Published online September 28, 2021 https://doi.org/10.4014/jmb.2104.04026
Copyright © The Korean Society for Microbiology and Biotechnology.
Suppression of Fusarium Wilt Caused by Fusarium oxysporum f. sp. lactucae and Growth Promotion on Lettuce Using Bacterial Isolates
Dil Raj Yadav, Mahesh Adhikari, Sang Woo Kim, Hyun Seung Kim, and Youn Su Lee*
Department of Applied Plant Sciences, Interdisciplinary Program in Smart Agriculture, Kangwon National University, Chuncheon 24341, Republic of Korea
Correspondence to:Youn Su Lee, younslee@kangwon.ac.kr
Abstract
This study was carried out to explore a non-chemical strategy for enhancing productivity by employing some antagonistic rhizobacteria. One hundred eighteen bacterial isolates were obtained from the rhizospheric zone of various crop fields of Gangwon-do, Korea, and screened for antifungal activity against Fusarium wilt (Fusarium oxysporum f. sp. lactucae) in lettuce crop under in vitro and in vivo conditions. In broth-based dual culture assay, fourteen bacterial isolates showed significant inhibition of mycelial growth of F. oxysporium f. sp. lactucae. All of the antagonistic isolates were further characterized for the antagonistic traits under in vitro conditions. The isolates were identified on the basis of biochemical characteristics and confirmed at their species level by 16S rRNA gene sequencing analysis. Arthrobacter sulfonivorans, Bacillus siamensis, Bacillus amyloliquefaciens, Pseudomonas proteolytica, four Paenibacillus peoriae strains, and Bacillus subtilis were identified from the biochemical characterization and 16S rRNA gene sequencing analysis. The isolates EN21 and EN23 showed significant decrease in disease severity on lettuce compared to infected control and other bacterial treatments under greenhouse conditions. Two bacterial isolates, EN4 and EN21, were evaluated to assess their disease reduction and growth promotion in lettuce in field conditions. The consortium of EN4 and EN21 showed significant enhancement of growth on lettuce by suppressing disease caused by F. oxysporum f. sp. lactucae respectively. This study clearly indicates that the promising isolates, EN4 (P. proteolytica) and EN21 (Bacillus siamensis), can be commercialized and used as biofertilizer and/or biopesticide for sustainable crop production.
Keywords: Antifungal activity, biocontrol, Fusarium oxysporum f. sp. lactucae, rhizobacteria
Introduction
Naturally occurring bacteria have been suggested as a replacement for supplements and chemical pesticides to control plant diseases [1]. Various species of bacteria have been focused on because of their root-colonizing capacity as well as their catabolic adaptability and production of metabolites with antibacterial and antifungal efficacy [2]. Several species of soil and seed-borne plant pathogenic fungi, such as
Control of all these phytopathogens is hugely based on genetic resistance in the host plant, use of synthetic pesticides, and environmental factors as well as management of the plant pesticides [4].
However, the use of chemical pesticides leads to inequality in the microbial community and may create new strains of resistant pathogens against beneficial microorganisms [5]. Therefore, the beneficial effects of rhizobacteria towards various phytopathogens can be explored for sustainable crop production [6, 7].
Soil bacteria and fungi possess some vital processes such as nitrogen fixation, nutrient mineralization and mobilization, decomposition and denitrification. The motility of bacteria has great impact on their ability to thrive in soil and colonization in the beginning phases where movement and attachment to the root surface are crucial [8]. Hence, essential identification of bacteria mostly involves the determination of colony morphology, catalase and oxidase testing, Gram staining, Voges-Proskauer tests (IMViC), and utilization of sugars (IMViC) [9].
Characterization of bacterial isolates via biochemical assay using organic manure and solid waste degradation was carried out based on IMViC and catalase oxidase through testing [10].
Applying environment friendly biocontrol agents is a specific and natural way to control plant pathogens and increase crop production [11].Several studies have been reported regarding the suppression of plant pathogens under in vitro and in vivo conditions using
The main objectives this study were to (i) isolate the potential antagonistic rhizobacteria from various sources and (ii) suppress Fusarium wilt caused by
Materials and Methods
Soil Sample Collection and Isolation of Bacterial Isolates
In total, 25 soil samples were collected from Chuncheon (37°56'21.69'' N, 127°46'55.30'' E), Hongcheon (37°41'46.74'' N, 127°54'19.01'' E), and Hwacheon (38°06'37.99'' N, 127°41'14.02'' E) in Gangwon-do, Korea, during April to June 2014. Soil sample collection was performed following the method of Harley and Waid (1975)[18]. Soil samples were dug about 2-3 inches from the immediate vicinity of rice (
Fungi and Culture Conditions
The fungal pathogen (
Screening of Bacteria Antagonistic to Fusarium oxysporum f. sp. lactucae
All bacterial isolates were screened for antagonistic activity against tested fungal plant pathogens using dual plate culture technique [20] with slight modification. Briefly, 6 mm mycelial plugs of actively growing pathogens were placed on the center of plates (150 mm diameter) containing PDA medium, and then eight sterilized paper discs (6 mm diameter) were placed equidistantly about 1.5 cm from the edge of the same plate. Suspensions of 10 μl of each bacterial isolate were inoculated in the paper discs and incubated for 7 days at 28°C until the fungus in the control plate covered the edge of the plate. The plates without bacterial inoculation and containing fungal plugs only were considered as control. Scoring technique was applied to measure the mycelial growth inhibition of pathogen. Zero (0) indicates the bacterial isolates fully covered with hyphae of fungi, one (1) indicates the fungal hyphae at the edge of the bacterial colony and, two (2) indicates a clear fungal mycelial inhibition zone around the bacterial colony [21]. The isolates with a score of 2 were only considered as antagonists and were selected for further evaluation of antagonistic properties by different methods.
Determination of Percentage Inhibition of Mycelial Growth of Fungal Pathogens
Antagonistic efficacy of screened bacterial isolates was further evaluated by dual culture technique. Briefly, 6 mm mycelial plugs of actively growing pathogens were placed on the center of Petri dishes (90 mm) containing 25 ml PDA-TSA (1:1 v/v), and then three sterilized paper discs (6 mm) were placed equidistantly about 1.5 cm from the edge of the same plate. Each paper disc was inoculated with a 10 μl freshly grown bacterial suspension at the concentration of 108 CFU/ml. The plates without bacterial inoculation and containing fungal plug only were considered as control. The test was done in triplicate. The antagonistic effect was determined by measuring the size of the inhibition zones and the radial growth of fungal mycelium. The percent inhibition of growth over control was calculated using this formula:
Inhibition of Fungal Mycelium Proliferation by Broth Culture Assay
One milliliter of 48-hour-old bacterial culture and two discs of 6 mm of test fungi were inoculated in 50 ml of PDB and TSB (1:1 v/v) in a conical flask of 250 ml at 28°C on a rotary shaker at 150 rpm (replications were made thrice per isolate). Control represents the broth inoculated with fungus only. The differences in dry weights between the bacterium treated and the control cultures were recorded by passing dual cultures grown for 7 days through pre-weighed filter paper. The filter papers were dried for 24 h at 70°C and weighed. The experiment had a completely randomized design with three replications. The reduction in weight of the test fungi was calculated using this formula in percentage [22]:
Reduction in weight (%) = (W1-W2)/W1×100,
where, W1 represents the weight of the test fungus in control flasks and W2 with the bacterial antagonists.
Elucidation of Antagonistic Traits
Chitinase activity. Qualitative estimation of chitinase was carried out in chitin agar plates prepared and amended with 2% phenol red and isolates (10 μl) were inoculated into wells. The plates were incubated for 120 h at 25-29°C and the chitinase activity was indicated as clear halos around the inoculated holes. The magnitude of the activity was calculated by measuring the diameter of the zones. The test was repeated in triplicate for each isolate [23].
Protein hydrolysis. Skim milk agar plates (skim milk 100 g, peptone 5 g, agar 15 g, distilled water 1,000 ml) were prepared and inoculated with pure bacterial culture into wells. The inoculated plates were incubated at 28°C for 48 h, and the plates were observed for clear zones around the wells [24].
Pectinase and cellulase production. To determine pectinase and cellulose production, the media were prepared by adding 1% pectin and cellulose in basal medium (NaNO3 1 g, K2HPO4 1 g, KCl 1 g, MgSO4.7H2O 0.5 g, yeast extract 0.5 g, glucose 1 g, distilled water 1,000 ml, agar 15 g). Ten microliters of the bacterial cell suspension was inoculated into the wells made on the medium and incubated for 5 days at 28°C. Gram’s iodine solution (3%) was poured in the pectin and cellulose agar media and zones of clearance were observed against the dark blue background. A clear zone against the blue background indicated that the bacteria were positive for pectinase and cellulase production. The magnitude of the activity was calculated by measuring the diameter of the zones. The test was repeated in triplicate for each isolate [24].
Elucidation of Plant Growth-Promoting Traits
Hydrogen cyanide (HCN) production. Nutrient agar amended with 4.4 g/l glycine and bacteria was streaked (log phase) onto plates. A Whatman filter paper No. 1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed at the top of the plates which were then sealed with parafilm and incubated at 35-37°C for 4days. Development of orange to red color indicated HCN production [25].
Hydrolysis of starch. Starch agar plates (peptone 5 g, beef extract 3 g, soluble starch 0 g, agar 15 g, distilled water 1,000 ml) were prepared and inoculated with pure bacterial culture and incubated at 25-29°C for 48 h. After incubation, iodine (3%) was poured onto the plates. Formation of a blue-black color due to starch-iodine complex in the unutilized places of starch in the agar plates was indicated. Starch hydrolysis by the bacteria via production of amylase was indicated by a clear halo zone surrounding the bacterial colony on the starch agar medium. The test was repeated thrice for each culture and recorded [24].
Siderophore production. Siderophore production by bacterial isolates was detected by the universal method of Schwyn and Neilands (1987) [26] using chrome azurol S (CAS) media. CAS agar plates were prepared and inoculated with the 10 μl of exponentially growing test bacterial culture (0.5 OD at 620 nm) and incubated at 28°C for 7 days. Development of a yellow-orange halo around the colony was considered as positive for siderophore production. The test was repeated thrice for all the cultures and siderophore production efficiency (SPE) was calculated by the following formula:
Ammonia production. Bacterial isolates (50 μl of bacterial cell suspension) were grown in 30 ml peptone water broth (4%) for five days at 25-29°C. Two milliliters of culture supernatant was mixed with 1 ml Nessler’s reagent and a volume of this mixture was increased to 8.5 ml by addition of ammonia-free distilled water. Development of yellow-to-brown color indicated ammonia production, and the optical density was measured at 450 nm using a spectrophotometer. The concentration of ammonia was estimated using the standard curve of ammonium sulphate in the range of 0.1-1.0 μmole/ml.
Indole acetic acid production (IAA). IAA production was estimated using the method described by Bric
Ten percent exponentially grown bacterial strain culture was inoculated in 100 ml NB (or 50 μl cell suspension in 5 ml of the sterile peptone yeast extract broth (peptone 10 g, beef extract 3 g, NaCl 5 g), with varying concentrations of L-tryptophan ranging from 0 to 500 μg/ml in a 15-ml tube. The broth (2 ml) was collected at 24, 48, and 72 h and centrifuged at 2,700 g for 15 min followed by assay for quantitative measurement of IAA. Then, 1 ml of the cell-free supernatant was mixed vigorously with 1 ml Salkowsky’s reagent (1 ml of 0.5M FeCl3 in 50 ml of 35% HClO4-perchloric acid) along with two drops of orthophosphoric acid and the assay system was kept at room temperature (25-29°C) in dark for 20 min till pink color developed (in a 2-ml Eppendorf tube). Optical density was measured spectrophotometrically at 535 nm. The concentration of IAA in each sample was determined from the standard curve of IAA with the standards prepared in the range of 10-100 μg/ml of IAA [28].
Phosphate solubilization. Phosphate solubilization activity of the selected rhizobacterial isolates was detected by means of plate assay using Pikovskaya (PVK) agar, which results in a clear halo formation. A pure colony from a fresh culture of each isolate was inoculated at four equidistant points into each of the PVK-agar media using a sterile needle. The diameter of the clear halo zone was observed after 12 days of incubation at 28°C. Control plates were inoculated with sterile tryptic soy broth (TSB) only. The diameters of the colony and clearing zones around the colonies were measured. All the tests were replicated thrice. The solubilization index of the isolates was calculated with the formula given below:
Zinc solubilization. The selected antagonistic bacterial isolates were inoculated into modified PVK medium (ingredients g/l), (glucose 10.0 g, ammonium sulphate 1.0 g, potassium choloride 0.2 g, dipotassium hydrogen phosphate 0.2 g, magnesium suphate 0.1 g, yeast 0.2 g, distilled water 1,000 ml, pH 7.0) containing 0.1% insoluble zinc compounds ( ZnO, ZnCO3, and ZnS). The test organisms were inoculated on these media and incubated at 28°C for 7 days. The diameters of the clear zone around the colonies were measured. All the tests were replicated thrice. The solubilization index of the isolates was calculated with the formula given below:
Molecular identification and phylogenetic analysis. For the extraction of DNA, the bacterial cells were harvested from 10 ml overnight culture and pellets were lysed in 1 ml lysis buffer (25% sucrose, 20 mM EDTA, 50 mM Tris-HCl and 5 mg/ml-1 of lysozyme). Chromosomal DNA was extracted following the standard procedure [29]. Universal primers 27F and 1492R were used to amplify the 16 rRNA using PCR [30]. The PCR was carried out in a thermocycler using 35 amplification cycles at 94°C (45 sec), 55°C (60 sec), and 72°C (60 sec) with a final extension for 7 min at 72°C. Products obtained from the PCR were purified by using a Montage PCR Clean-Up Kit (Millipore, USA). Universal primers, 518F and 800R (Macrogen, Korea) were used to sequence the purified PCR products of approximately 1,400 bp through a big Dye Terminator Cycle Sequencing Kit v.3.1 (Applied BioSystems, USA). An Applied BioSystems model 3730XL automated DNA sequencing system (Applied BioSystems) at Macrogen Inc. Seoul, Korea was used to resolved the sequencing products. The sequences were compared using the NCBI (National Center for Biotechnology Information) BLAST (Basic Local Alignment Search Tool) program (http://www.ncbi.nlm.nih.gov/Blast) for identification of the isolates. All positions containing gaps and missing data were eliminated from the dataset. Best hit sequences were downloaded in FASTA format from the NCBI database to construct a phylogenetic tree using MEGA 6 software [31].
Disease suppression by rapid radicle assay. The bacterial isolates were cultured in TSB (tryptic soy broth) with shaking at 150 rpm at 28°C for 48 h for bacterial suspensions. Seeds of lettuce were surface sterilized with 5%sodium hypochlorite for 20 min, washed thrice with sterile distilled water and kept in Petri dishes with moist filter paper for 3-4 days at 25°C in darkness for germination. Uniformly germinated seeds were soaked in the bacterial suspensions (108 cells/ml-1) of isolates. The treated seeds of lettuce were placed on the margins of actively growing mycelia of
Greenhouse and Field Evaluations
Preparation of fungal pathogen inoculum and inoculation technique. The pure culture of targeted fungal pathogen,
Experimental design and treatments. The greenhouse and field experiments were set up in RCB (Randomized Complete Block) designs with five replications. Ten bacterial isolates along with positive and negative controls were evaluated for their growth promotion and disease suppression activities on lettuce under greenhouse conditions. For growth promotion experiments, the positive control was maintained by mixing the autoclaved soil with chemical fertilizer (18 N: 7 P: 9 K) of 1 kg/1,000 m2 and uninoculated soil was treated as negative control. Three controls, infected with pathogen, non-infected or healthy, and positive (sprayed with 0.2% solution of Mancozeb 75% WP twice at intervals of seven days) were used in the disease evaluation experiments. Two potential bacterial isolates were tested under field conditions for their growth promotion and disease suppression activities on lettuce.
Observations. Disease severity (S) for Fusarium wilt of lettuce was estimated (after 5 and 8 weeks of transplanting, respectively), as a wilting percent using the rating scale in which infected plants were classified according to numerical grades ranging from 0 to 4 as follows: 0 = healthy, 1 = ≤ 25% of plant leaflets are yellow and of vascular root bundles are dark brown, 2 = ≥ 26-50% of plant leaflets are yellow and of vascular root bundles are dark brown, 3 = ≥ 50-75% of plant leaflets are yellow and of vascular root bundles are dark brown and 4 = ≥ 76-100% of plant leaflets are yellow and of vascular root bundles are dark brown.
where, A, B, C, and D are the number of plants corresponding to the numerical grades 1, 2, 3, and 4, respectively, and 4T is the total number of plants (T) multiplied by the maximum discoloration grade 4, where T = A + B + C + D. Reduction percentage was calculated using the formula of Guo
Statistical analysis. One-way analysis of variance (ANOVA) was applied to analyze the data from in vitro and to determine the significance of treatment effects. The percent data and data set having value zero (0) were transferred into arcsine square root transformation before further statistical analysis to improve the homogeneity of the variance of the data. Where the F values were significant, post hoc comparisons of means were made using Duncan’s multiple range test (DMRT) at the 0.05 probability level. All statistical analyses were done using CROPSTAT version 7.2.3 [34].
Results
Culturable Bacteria in the Rhizosphere and Endosphere
Bacteria were obtained both from the rhizospheric portion of various crop plants as well as the root interior of oat plants. Ninety-five bacteria were isolated from rice, maize, barley, sesame and soybean rhizospheric soil; and 23 were recovered from oat root interiors (Table S1). The general isolation frequency was 3.37. The isolation frequency in rhizospheric soil samples of rice, sesame, soybean, maize and oat was 4.86, 4.33, 4.00, 4.00, and 2.50, respectively. The lowest isolation frequency (2.30) was recorded in oat root samples and the highest number of isolates was recorded from rice rhizospheric soil samples.
Screening of Antagonistic Bacteria
Out of the 118 isolates tested, 20 isolates showed antagonism against all the test pathogens. The number of isolates with a score of 2 was 14 against
In Vitro Inhibition of F. oxysporum f. sp. Lactucae by Bacterial Isolates
All the screened bacterial isolates possessed inhibition against the tested pathogenic fungi. The highest inhibition was recorded by EN21 and OR7 (Table 1 and Fig. 1). Moreover, all tested bacterial isolates showed biomass reduction in all tested fungi with varied rate of reduction. The mycelial biomass of all tested fungi was reduced to the highest degree in dual culture broths inoculated with bacterial isolate EN21 (Fig. 2).
-
Table 1 . Antagonistic efficacy of rhizobacterial isolates against
F. oxysporum f. sp.lactucae in dual culture assay..Isolates Fungal pathogens F. oxysporum f. sp.lactucae RR8 56.1d (5.0d-e) RR12 52.9e (4.3d-f) RR26 54.5de (4.7de) RR33 49.8f (3.3f) RR34 55.7d (5.3d) MR3 53.7de (5.0de) MR19 62.0c (3.7ef) OR7 66.3a (7.7c) OR19 63.1bc (5.7d) EN4 0.0g (0.0g) EN18 63.9a-c (8.7bc) EN20 65.5ab (8.3c) EN21 66.3a (10.0a) EN22 65.4ab (8.7bc) EN23 65.5ab (9.7ab) Control 0.0g (0.0g)
-
Figure 1. Growth promotion of
Fusarium oxysporum f. sp.lactucae by selected rhizobacterial isolates in dual culture assay. (A) Control; (B) RR8; (C) RR12; (D) RR26; (E) RR33; (F) RR34; (G) MR3; (H) MR19; (I) OR7; (J) OR19; (K) EN4; (L) EN18; (M) EN20; (N) EN21; (O) EN22 and (P) EN23.
-
Figure 2. Reduction in mycelial dry weight biomass of
Fusarium oxysporum f. sp.lactucae due to antagonism of rhizobacterial isolates. Values with different lowercase letters indicate significant differences atp ≤ 0.05. Error bars indicate the standard error of three replicates.
Elucidation of Antagonistic Traits
Fifteen bacterial isolates were tested for antagonistic traits viz., chitinase, protease, pectinase and cellulase production. Clearing of plates containing colloidal chitin as a sole carbon source by the bacterium around the colony was used to measure chitin hydrolysis. All isolates, except RR33 and EN4, showed strong chitinolytic activity (Table 2, Fig. S1). The isolates RR34 and EN4 were weak producers of chitinase. Starch hydrolysis was observed via zones of starch hydrolysis through the production of α-amylase. Clearing of starch agar plates containing starch as a sole source of carbon by the bacterium around the colony was used to measure starch hydrolysis. Out of 15 isolates, 13 isolates were producers of α-amylase. The isolates RR34 and EN4 demonstrated negative response to starch hydrolysis (Table 2, Fig. S2). Clearing of skim milk agar plates containing skim milk as a sole source of protein by the bacterium around the colony was used for qualitative detection of protease production. Out of 15 isolates, 14 isolates demonstrated positive response to protein hydrolysis. The isolate RR34 was found negative with regard to production of protease (Table 2, Fig. S3). Cellulose degradation was observed via zones of cellulose hydrolysis through the production of cellulase. Clearing of agar plates containing cellulose powder as a sole source of cellulose by the bacterium around the colony was used. Out of 15 isolates, 14 isolates demonstrated positive response to cellulose degradation. The isolate EN4 was found negative for the production of cellulase (Table 2, Fig. S4).
-
Table 2 . Antagonistic traits of selected antagonistic bacterial isolates..
Isolates Hydrolytic enzymes HCN production Siderophore production Chitinase Endozymes Protease Cellulase Pectinase α-amylase Catalase Oxidase RR8 ++ +++ ++ ++ - - +++ ++ ++ RR12 ++ +++ ++ ++ - + +++ ++ ++ RR26 +++ +++ +++ +++ - + +++ +++ +++ RR33 + +++ ++ ++ - + + + + RR34 - + - - - + +++ + + MR3 ++ +++ ++ ++ - +++ +++ ++ ++ MR19 ++ +++ ++ ++ - +++ +++ +++ +++ OR7 +++ +++ +++ +++ - +++ +++ +++ +++ OR19 +++ +++ +++ +++ - +++ +++ +++ +++ EN4 +++ - - - - +++ + ++ ++ EN18 +++ +++ +++ +++ - +++ +++ ++ ++ EN20 +++ +++ +++ +++ - +++ +++ ++ ++ EN21 +++ +++ +++ +++ - +++ +++ ++ ++ EN22 +++ +++ +++ +++ - +++ +++ + ++ EN23 +++ +++ +++ +++ - +++ +++ ++ ++ +++ = high, ++ = medium, + = low, - = negative producer..
Growth-Promoting Trait Elucidation of Plant
The formation of yellow-to-orange halos was indicative of siderophore production. All tested isolates, except RR8, were positive for siderophore production (Table 2, Fig. S5).
Bacterial isolates were grown in peptone water broth for detection of ammonia production. Tubes showing faint yellow indicated a small amount of ammonia production, and deep yellow to brownish color indicated a maximum amount of ammonia production. Out of 15 isolates, 12 isolates were positive for ammonia production (Table 3). The isolates RR8, RR12 and RR33 showed negative response to ammonia production. The production of ammonia by the isolates EN4 and EN21 was more evident than the other isolates (Table 3). Maximum ammonia produced by the isolates EN4 and EN21 was 5.7 and 5.6 μmole/ml, respectively (Fig. 3 and Table 3).
-
Table 3 . Growth promoting traits of selected antagonistic bacterial isolates..
Isolates IAA† NH3 production† Phosphate solubilizationα Zinc solubilizationα RR8 - - + + RR12 - - + + RR26 - ++ - - RR33 - - - + RR34 - + - ++ MR3 - ++ - +++ MR19 - ++ - + OR7 + ++ - - OR19 - ++ - ++ EN4 ++ +++ +++ +++ EN18 - ++ - - EN20 - ++ - - EN21 ++ +++ ++ - EN22 - ++ - - EN23 - ++ + +++ †+++ = strong, ++ = medium, + = weak and - = no production of IAA and NH3;.
α+++ = strong, ++ = medium, + = weak and - = no solubilization of phosphate and zinc..
-
Figure 3. Ammonia production by bacterial isolates. (A) Control; (B) RR8; (C) RR12; (D) RR26; (E) RR33; (F) MR3; (G) MR19; (H) OR7; (I) OR19; (J) EN4; (K) EN18; (L) EN20; (M) EN21; (O) EN23 and (P) RR34.
It was observed that out of 15 isolates, only three isolates OR7, EN4 and EN21 could produce IAA only when L-tryptophan was supplemented in the medium. IAA production by the isolates was determined after 72 h of incubation and maximum IAA produced was 8.6 μg/ml by the isolate EN4 when L-tryptophan concentration in the medium was maximum (500 μg/ml) (Fig. 4). In the growth medium with absence of L-tryptophan, IAA was not detected in any of the three isolates even after 72 h (Fig. 4 and Table 3). This shows that there is a direct correlation between IAA production and supplemented L-tryptophan in the medium.
-
Figure 4. Indole acetic acid produced by selected bacterial isolates at 72 h of incubation in different concentrations of L-tryptophan supplemented in nitrogen free broth. Error bar denotes the standard error of three replicates.
The bacterial isolates that showed zones of clearance on PVK agar media were considered as phosphate solubilizers and the phosphate solubilization index of all 15 bacterial isolates is shown in Table 3. Out of 15 isolates, five isolates demonstrated phosphate solubilization activity. The isolates RR8, RR12 and EN23 showed low solubilization efficiency while the isolates EN21 and EN4 demonstrated medium and high solubilization efficiency, respectively. The solubilization index of EN4 and EN21 was 4.0 and 2.2, respectively. Quantitative estimation of solubilized phosphate by potent bacterial isolates, EN4 and EN21, was done by PVK broth method. The amount of solubilized phosphate by the isolates EN4 and EN21 was 376.0 and 173.3 mg/l, respectively (Table 3, Fig. S6).
For zinc solubilization, the results showed that only nine isolates out of 15 isolates could form clearing zones in plate assay. Zinc solubilization potential varied among bacterial isolates (Table 3). The isolate EN4 showed the highest potential of zinc solubilization both in zinc oxide and zinc carbonate-containing media. It produced a clear zone of 16.7 and 15.7 mm with solubilization index of 3.4 and 3.2 in plates containing zinc oxide and zinc carbonate, respectively (Table 3, Fig. S7). HCN production by the bacterial isolates was observed as a change in color of the filter paper from yellow to orange brown. None of the tested isolates was found positive to HCN production (Table 2, Fig. S8).
Molecular Identification of the Bacterial Isolates
The molecular analysis revealed that 15 isolates belonged to three groups, Firmicutes, Proteobacteria, and Actinobacteria (Fig. 5). Most of the antagonistic bacteria (13 isolates, 86.6% of total) belonged to the Firmicutes group. Phylogenetic analysis based on 16S rRNA gene sequences indicated that
-
Table 4 . Similarity scores between bacterial isolates and the highly matched type strain identified by neighbor-joining analysis..
Bacterial isolates Closest GenBank accession No. Closest GenBank taxa Similarity (%) RR8 (KU512890) AB073186 Paenibacillus peoriae 99.5 RR12 (KU512891) AB073186 Paenibacillus peoriae 99.0 RR26 (KU512892) AF235091 Arthrobacter sulfonivorans 98.6 RR33 (KU512893) AB073186 Paenibacillus peoriae 99.1 RR34 (KU512894) AB073186 Paenibacillus peoriae 99.1 MR3 (KU512895) AB271744 Bacillus subtilis 99.7 MR19 (KU512896) AB271744 Bacillus subtilis 100.0 OR7 (KU512897) GQ281299 Bacillus siamensis 99.5 OR19 (KU512898) AB325583 Bacillus amyloliquefaciens 99.8 EN4 (KU512899) AJ537603 Pseudomonas proteolytica 99.0 EN18 (KU512900) GQ281299 Bacillus siamensis 99.5 EN20 (KU5129101) GQ281299 Bacillus siamensis 99.4 EN21 (KU5129102) GQ281299 Bacillus siamensis 99.4 EN22 (KU5129103) GQ281299 Bacillus siamensis 99.4 EN23 (KU5129104) GQ281299 Bacillus siamensis 99.4
-
Figure 5. Phylogenetic analysis of internal transcribed spacer regions (16S rRNA gene sequences) of rhizobacteria isolated from various places in Gangwon-do, Korea. MEGA 6 software was used to construct the phylogenetic tree. Boldface indicates the sequences obtained in this study. Numerical values (>50) on branches indicates the percentage of 1,000 bootstrap replicates that support the branch. The scale bar expressed the number of changes per site.
Suppression of F. oxysporum f. sp. lactuace and Growth Promotion on Lettuce under In Vitro and In Vivo Conditions
The growth of lettuce seedlings with and without bacterial inoculation, based on root and shoot length and dry weight of whole plant, after 14 days of treatments, is presented in Table 5. The seed inoculations with bacterial strains increased the mentioned growth parameters over negative control and the increment was significant (
-
Table 5 . Efficacy of bacterial isolates on lettuce seedling growth by test tube method in vitro..
Isolates Shoot length (cm) Root length (cm) Seedling weight (mg/seedling) Fresh Dry RR8 10.57d 11.60ef 775.77g 45.03d RR12 10.43d 10.80fg 583.03k 40.10d RR26 9.47e 9.80h 552.70l 32.02e RR33 7.40f 10.07gh 549.87l 30.69e MR3 9.43e 11.00f 608.35j 43.36d MR19 9.60e 10.90f 630.43i 43.03d OR7 10.63d 12.47c-e 761.90g 45.20d OR19 10.53d 12.43c-e 707.68h 43.03d EN4 12.53b 13.33b 1235.90b 74.50a EN18 10.67d 12.33c-e 873.53f 55.13c EN20 10.97d 12.27c-e 1035.68e 54.23c EN21 12.20bc 13.03bc 1164.36c 65.35b EN22 11.67c 12.03de 882.70f 45.36d EN23 11.67c 12.70b-d 1128.36d 66.37b Positive Control 13.20a 15.07a 1321.83a 75.34a Negative Control 6.37g 8.30i 533.17m 30.70f Data are means of 10 replications..
Values with different alphabetic superscripts in the same column are significantly different at p ≤ 0.05 levels according to Duncan’s multiple range test..
-
Figure 6. Efficacy of bacterial isolates on lettuce seedling growth by test tube method. (A) Negative control; (B) Positive control; (C) RR8; (D) RR12; (E) RR26; (F) RR33; (G) MR3; (H) MR19; (I) OR7; (J) OR19; (K) EN4; (L) EN18; (M) EN20; (N) EN21; (O) EN22, and (P) EN23.
-
Figure 7. Disease occurrence caused by
Fusarium oxysporum f. sp.lactucae on radicles of lettuce seeds (cv.Jukchima ) treated with bacterial strains. Germinated lettuce seeds treated with distilled water (control) or bacterial suspensions for 2 h were placed on to the margin of actively growing mycelia ofFusarium oxysporum f. sp.lactucae on water agar containing 0.02% glucose for 7 days. Lowercase letters expressed the significant differences atp ≤ 0.05. The experiment was conducted with four replications of 5 seeds each. Square root transformed data were used for data analysis.
The results of the greenhouse experiment revealed that inoculation with bacterial isolates significantly promoted the growth of lettuce plants over negative control. However, the rate of enhancement varied with bacterial strains. Of tested isolates, isolate EN4 extensively increased all the growth attributes by recording 44.80 cm plant height, 1428.67 cm2 leaf area per plant, 38.40 chlorophyll content SPAD value, 1.80 g of root dry weight per plant, 6.35 g of shoot dry weight per plant and 20.50 cm root length (Table 6 and Fig. 8). The results were significantly higher than negative control and most of the bacterial isolates. The results revealed that the effects of isolates EN4 and EN21 were comparable to chemical fertilizer though all the crop attributes were significantly higher in plants treated with chemical fertilizer. Moreover, EN21 showed highest suppression (66.11%) of tested pathogen under greenhouse conditions (Table 7). The results also showed that plants inoculated with any of the tested bacterial isolates significantly reduced wilting percentage (Fig. 7). The highest disease severity reduction was observed with isolate EN21 and then by EN23. The reductions in disease severity by these two isolates were 66.11 and 60.68%, respectively (Table 7). The lowest reductions were produced by isolates EN4 and RR8 (26.21 and 32.06%, respectively). The isolate EN21 caused a 140.5% increment in dry shoot weight over infected control by reducing wilting (Table 7).
-
Table 6 . Effects of bacterial isolates on growth parameters of lettuce in soil treatments under greenhouse conditions..
Isolates Plant height (cm) Leaf area (cm2/plant) Chlorophyll content (SPAD value) Fresh weight (g/plant) Dry weight (g/plant) Root length (cm) Root Shoot Root Shoot RR8 34.17fg 1235.67h 31.03gh 8.47f 51.98g 0.85g 4.12ef 15.53g MR19 34.13fg 1230.33h 30.77h 8.44f 51.90g 0.82g 3.71f 15.53g OR7 35.57e 1330.00g 32.80ef 12.71e 53.48g 1.14e 4.69d 16.33f OR19 35.03ef 1327.00g 31.87fg 12.39e 53.22g 0.96f 4.58de 16.13fg EN4 44.80b 1428.67b 38.40b 18.73b 92.29b 1.80b 6.35a 20.50b EN18 35.47e 1346.33f 34.83d 13.52d 71.02d 1.29d 4.97cd 17.33de EN20 33.50g 1320.67g 33.47e 13.45d 62.01f 1.28d 4.71d 16.63ef EN21 42.67c 1415.67c 36.93c 18.51b 75.94c 1.72b 5.56b 18.50c EN22 35.87e 1362.67e 35.57d 14.64c 67.79e 1.50c 5.03cd 17.43d EN23 38.53d 1391.67d 35.13d 14.75c 71.32d 1.53c 5.37bc 18.30c Positive Control 51.63a 1530.00a 40.70a 20.70a 98.37a 2.87a 6.72a 21.47a Negative Control 31.17h 462.33i 28.50i 7.51g 41.43h 0.75h 2.85g 13.20h Data are means of five replications..
Values with different alphabetic superscripts in the same column are significantly different at p ≤ 0.05 levels according to Duncan’s multiple range test..
-
Table 7 . Effect of inoculation with rhizobacteria on development of Fusarium wilt and shoot dry weight on lettuce under greenhouse conditions..
Treatmentsa Disease severityb (%) Disease reduction (%) Shoot dry weight (g/plant) RR8 65.67bc 32.06 3.67g MR19 61.33c 36.56 3.67g OR7 45.33d 53.10 4.45e OR19 44.00de 54.47 4.47e EN4 71.33b 26.21 4.2f EN18 42.00de 56.54 4.85c EN20 44.00de 54.49 4.72d EN21 34.67fg 66.11 6.35a EN22 40.33def 58.27 4.93c EN23 38.00e-g 60.68 6.12b Chemical 32.67g 64.12 2.92h Non-infected Control - - 2.75i Infected Control 96.67a - 2.64j
-
Figure 8. Shoot growth promotion on lettuce by bacterial isolates under greenhouse conditions. (A) Negative control; (B) Positive control; (C) RR8; (D) MR19; (E) OR7; (F) OR19; (G) EN4; (H) EN18; (I) EN20; (J) EN21; (K) EN22; and (L) EN23.
Inoculation of plants with
-
Table 8 . Effect of inoculation with rhizobacteria on development of Fusarium wilt and shoot length of lettuce under field conditions..
Treatmentsa Shoot length (cm) Disease severityb (%) Disease reduction (%) EN21 85.17b 45.9b 44.91 EN4+21 94.83a 35.7cd 57.15 Chemical 74.83c 30.5d 63.39 Non-infected Control 64.67d - - Infected Control 28.17e 83.33a - aLettuce plants (cv. Jukchima) were treated by drenching the soil around root zone with the broth culture of bacterial isolates two times at an interval of seven days. Control plants (not infected and infected control) were treated with tap water and plants were sprayed with 0.2% solution of Mancozeb 80WP two times at an interval of seven days..
bDisease severity was recorded at 8 weeks after planting..
Data are means of five replications. Values with different alphabetic superscripts in the same column are significantly different at
p ≤ 0.05 levels according to Duncan’s multiple range test..
-
Figure 9. Effect of inoculation with rhizobacteria on development of Fusarium wilt and foliage yield of lettuce under field conditions.
Discussion
Soil microorganisms are regarded as an important and essential component of soil quality due to their crucial activities in many ecosystem processes [35, 36]. Rhizospheres have been frequently exploited as an excellent source of biocontrol agents, since they provide the frontline of defensive microorganisms for roots against the attack of soil-borne pathogens [37]. In this study, 20 antagonistic bacterial isolates out of 118 rhizobacterial isolates were screened with 13 fungal pathogens as targets. The antagonistic bacterial isolates exerted varied levels of antagonism against tested pathogens. Fluctuation in the spectrum of antifungal activity of bacteria is common [38]. In dual culture assays, isolates RR8, MR3, MR19, OR7, OR19, EN18, EN20, EN21, EN22, and EN23 showed maximum inhibition of radial growth of test pathogens. In this study, some bacterial isolates were found to be highly inhibitory of fungal growth whereas others showed only minor activity or no activity at all. The inhibition zone exhibited between the fungal pathogens and bacteria was expressed in the inhibition of fungal mycelium. Moreover, as the PDA medium used for the dual culture assay is rich in nutrients, competition might be excluded as the mode of action for these isolates [39]. The antifungal metabolites produced seems to vary among the bacterial isolates tested in this study. This suggests that the fungal mycelia might not only be inhibited by antibiosis but also by other antifungal metabolites such as siderophores, hydrogen ions and gaseous products including ethylene, hydrogen cyanide and ammonia [40]. In vitro broth-based dual cultures offer a better method for evaluation of antagonistic efficiency of the biocontrol agents as the liquid medium may provide a better environment to allow the antagonistic activities from all possible interacting sites. These results are in agreement with the findings of Ashwini and Srividya (2014) [41] who revealed that antagonistic bacteria,
This study revealed that some rhizobacterial isolates were capable of inhibiting a wide range of phytopathogens in controlled conditions. But, in most biocontrol investigations, a large number of antagonists are commonly isolated over a short period of time and screened in vitro for antagonistic activity and tests based on in vitro mycelial inhibition and root colonization do not always correlate with biocontrol efficacy under natural conditions [42]. However, little correlation exists between in vitro and in vivo antagonistic activity in general [43] and identification of promising field-effective bacteria, however, can be facilitated by greenhouse experiments [44]. The major bacterial genus identified in our studies was
In the present study,
Supplemental Materials
Acknowledgments
This study was conducted with the support of a research grant from Kangwon National University.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

Fig 6.

Fig 7.

Fig 8.

Fig 9.

-
Table 1 . Antagonistic efficacy of rhizobacterial isolates against
F. oxysporum f. sp.lactucae in dual culture assay..Isolates Fungal pathogens F. oxysporum f. sp.lactucae RR8 56.1d (5.0d-e) RR12 52.9e (4.3d-f) RR26 54.5de (4.7de) RR33 49.8f (3.3f) RR34 55.7d (5.3d) MR3 53.7de (5.0de) MR19 62.0c (3.7ef) OR7 66.3a (7.7c) OR19 63.1bc (5.7d) EN4 0.0g (0.0g) EN18 63.9a-c (8.7bc) EN20 65.5ab (8.3c) EN21 66.3a (10.0a) EN22 65.4ab (8.7bc) EN23 65.5ab (9.7ab) Control 0.0g (0.0g)
-
Table 2 . Antagonistic traits of selected antagonistic bacterial isolates..
Isolates Hydrolytic enzymes HCN production Siderophore production Chitinase Endozymes Protease Cellulase Pectinase α-amylase Catalase Oxidase RR8 ++ +++ ++ ++ - - +++ ++ ++ RR12 ++ +++ ++ ++ - + +++ ++ ++ RR26 +++ +++ +++ +++ - + +++ +++ +++ RR33 + +++ ++ ++ - + + + + RR34 - + - - - + +++ + + MR3 ++ +++ ++ ++ - +++ +++ ++ ++ MR19 ++ +++ ++ ++ - +++ +++ +++ +++ OR7 +++ +++ +++ +++ - +++ +++ +++ +++ OR19 +++ +++ +++ +++ - +++ +++ +++ +++ EN4 +++ - - - - +++ + ++ ++ EN18 +++ +++ +++ +++ - +++ +++ ++ ++ EN20 +++ +++ +++ +++ - +++ +++ ++ ++ EN21 +++ +++ +++ +++ - +++ +++ ++ ++ EN22 +++ +++ +++ +++ - +++ +++ + ++ EN23 +++ +++ +++ +++ - +++ +++ ++ ++ +++ = high, ++ = medium, + = low, - = negative producer..
-
Table 3 . Growth promoting traits of selected antagonistic bacterial isolates..
Isolates IAA† NH3 production† Phosphate solubilizationα Zinc solubilizationα RR8 - - + + RR12 - - + + RR26 - ++ - - RR33 - - - + RR34 - + - ++ MR3 - ++ - +++ MR19 - ++ - + OR7 + ++ - - OR19 - ++ - ++ EN4 ++ +++ +++ +++ EN18 - ++ - - EN20 - ++ - - EN21 ++ +++ ++ - EN22 - ++ - - EN23 - ++ + +++ †+++ = strong, ++ = medium, + = weak and - = no production of IAA and NH3;.
α+++ = strong, ++ = medium, + = weak and - = no solubilization of phosphate and zinc..
-
Table 4 . Similarity scores between bacterial isolates and the highly matched type strain identified by neighbor-joining analysis..
Bacterial isolates Closest GenBank accession No. Closest GenBank taxa Similarity (%) RR8 (KU512890) AB073186 Paenibacillus peoriae 99.5 RR12 (KU512891) AB073186 Paenibacillus peoriae 99.0 RR26 (KU512892) AF235091 Arthrobacter sulfonivorans 98.6 RR33 (KU512893) AB073186 Paenibacillus peoriae 99.1 RR34 (KU512894) AB073186 Paenibacillus peoriae 99.1 MR3 (KU512895) AB271744 Bacillus subtilis 99.7 MR19 (KU512896) AB271744 Bacillus subtilis 100.0 OR7 (KU512897) GQ281299 Bacillus siamensis 99.5 OR19 (KU512898) AB325583 Bacillus amyloliquefaciens 99.8 EN4 (KU512899) AJ537603 Pseudomonas proteolytica 99.0 EN18 (KU512900) GQ281299 Bacillus siamensis 99.5 EN20 (KU5129101) GQ281299 Bacillus siamensis 99.4 EN21 (KU5129102) GQ281299 Bacillus siamensis 99.4 EN22 (KU5129103) GQ281299 Bacillus siamensis 99.4 EN23 (KU5129104) GQ281299 Bacillus siamensis 99.4
-
Table 5 . Efficacy of bacterial isolates on lettuce seedling growth by test tube method in vitro..
Isolates Shoot length (cm) Root length (cm) Seedling weight (mg/seedling) Fresh Dry RR8 10.57d 11.60ef 775.77g 45.03d RR12 10.43d 10.80fg 583.03k 40.10d RR26 9.47e 9.80h 552.70l 32.02e RR33 7.40f 10.07gh 549.87l 30.69e MR3 9.43e 11.00f 608.35j 43.36d MR19 9.60e 10.90f 630.43i 43.03d OR7 10.63d 12.47c-e 761.90g 45.20d OR19 10.53d 12.43c-e 707.68h 43.03d EN4 12.53b 13.33b 1235.90b 74.50a EN18 10.67d 12.33c-e 873.53f 55.13c EN20 10.97d 12.27c-e 1035.68e 54.23c EN21 12.20bc 13.03bc 1164.36c 65.35b EN22 11.67c 12.03de 882.70f 45.36d EN23 11.67c 12.70b-d 1128.36d 66.37b Positive Control 13.20a 15.07a 1321.83a 75.34a Negative Control 6.37g 8.30i 533.17m 30.70f Data are means of 10 replications..
Values with different alphabetic superscripts in the same column are significantly different at p ≤ 0.05 levels according to Duncan’s multiple range test..
-
Table 6 . Effects of bacterial isolates on growth parameters of lettuce in soil treatments under greenhouse conditions..
Isolates Plant height (cm) Leaf area (cm2/plant) Chlorophyll content (SPAD value) Fresh weight (g/plant) Dry weight (g/plant) Root length (cm) Root Shoot Root Shoot RR8 34.17fg 1235.67h 31.03gh 8.47f 51.98g 0.85g 4.12ef 15.53g MR19 34.13fg 1230.33h 30.77h 8.44f 51.90g 0.82g 3.71f 15.53g OR7 35.57e 1330.00g 32.80ef 12.71e 53.48g 1.14e 4.69d 16.33f OR19 35.03ef 1327.00g 31.87fg 12.39e 53.22g 0.96f 4.58de 16.13fg EN4 44.80b 1428.67b 38.40b 18.73b 92.29b 1.80b 6.35a 20.50b EN18 35.47e 1346.33f 34.83d 13.52d 71.02d 1.29d 4.97cd 17.33de EN20 33.50g 1320.67g 33.47e 13.45d 62.01f 1.28d 4.71d 16.63ef EN21 42.67c 1415.67c 36.93c 18.51b 75.94c 1.72b 5.56b 18.50c EN22 35.87e 1362.67e 35.57d 14.64c 67.79e 1.50c 5.03cd 17.43d EN23 38.53d 1391.67d 35.13d 14.75c 71.32d 1.53c 5.37bc 18.30c Positive Control 51.63a 1530.00a 40.70a 20.70a 98.37a 2.87a 6.72a 21.47a Negative Control 31.17h 462.33i 28.50i 7.51g 41.43h 0.75h 2.85g 13.20h Data are means of five replications..
Values with different alphabetic superscripts in the same column are significantly different at p ≤ 0.05 levels according to Duncan’s multiple range test..
-
Table 7 . Effect of inoculation with rhizobacteria on development of Fusarium wilt and shoot dry weight on lettuce under greenhouse conditions..
Treatmentsa Disease severityb (%) Disease reduction (%) Shoot dry weight (g/plant) RR8 65.67bc 32.06 3.67g MR19 61.33c 36.56 3.67g OR7 45.33d 53.10 4.45e OR19 44.00de 54.47 4.47e EN4 71.33b 26.21 4.2f EN18 42.00de 56.54 4.85c EN20 44.00de 54.49 4.72d EN21 34.67fg 66.11 6.35a EN22 40.33def 58.27 4.93c EN23 38.00e-g 60.68 6.12b Chemical 32.67g 64.12 2.92h Non-infected Control - - 2.75i Infected Control 96.67a - 2.64j
-
Table 8 . Effect of inoculation with rhizobacteria on development of Fusarium wilt and shoot length of lettuce under field conditions..
Treatmentsa Shoot length (cm) Disease severityb (%) Disease reduction (%) EN21 85.17b 45.9b 44.91 EN4+21 94.83a 35.7cd 57.15 Chemical 74.83c 30.5d 63.39 Non-infected Control 64.67d - - Infected Control 28.17e 83.33a - aLettuce plants (cv. Jukchima) were treated by drenching the soil around root zone with the broth culture of bacterial isolates two times at an interval of seven days. Control plants (not infected and infected control) were treated with tap water and plants were sprayed with 0.2% solution of Mancozeb 80WP two times at an interval of seven days..
bDisease severity was recorded at 8 weeks after planting..
Data are means of five replications. Values with different alphabetic superscripts in the same column are significantly different at
p ≤ 0.05 levels according to Duncan’s multiple range test..
References
- Bano N, Musarrat J. 2003. Characterization of a new
Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent.Curr. Microbiol. 46 : 324-328. - Nelson EB, Maloney AP. 1992. Molecular approaches for understanding biological control mechanisms in bacteria: studies of interaction of
Enterobacter cloacae withPythium ultimum .Can. J. Plant Pathol. 14 : 106-14. - Armstrong GM, Armstrong JK. 1981. Formae speciales and races of
Fusarium , pp. 391-399.In: Nelson PE, Toussoun TA, Conk RJ (eds), . The Pennsylvania State University Press, University Park.Fusarium : diseases, biology and taxonomy - Ulloa M, Hanlin R. 1993. Plant disease control, pp. 448.
In: Strange R (ed),Plant Disease Control: Towards Environmentally Acceptable Methods , 1st ed. Chapman and Hall, New York. - Shanmugam V, Kanoujia N. 2011. Biological management of vascular wilt of tomato caused by
Fusarium oxysporum f. sp.lycopersici by plant growth-promoting rhizobacterial mixture.Biol. Control. 57 : 85-93. - Cavender ND, Atiyeh RM, Knee M. 2003. Vermicompost stimulates mycorrhizal colonization of roots of
Sorghum bicolor at the expense of plant growth.Pedobiologia (Jena) 47 : 85-89. - Jetiyanon K, Kloepper JW. 2002. Mixtures of plant growth-promoting rhizobacteria for induction of systemic resistance against multiple plant diseases.
Biol. Control 24 : 285-291. - Turnbull G a, Morgan JAW, Whipps JM, Saunders JR. 2001. The role of motility in the in vitro attachment of
Pseudomonas putida PaW8 to wheat roots.FEMS Microbiol. Ecol. 35 : 57-65. - Dubey RC, Maheshwari DK. 2005. Enhancement of collar rot in sunflower caused by
Sclerotinia rolfsii byPseuodomonas .Indian Phytopathol. 58 : 17-24. - Zaved HK, Rahman MM, Rahman MM, Rahman A, Arafat SMY, Rahman MS. 2008. Isolation and characterization of effective bacteria for solid waste degradation for organic manure.
KMITL J. Sci. Tech. 8 : 44-55. - Whipps JM. 1997. Ecological considerations involved in commercial development of biological control agents for soil-borne diseases, pp. 525-546.
In: van Elsas JD, Trevors, JT Wellington EMH (eds),Modern soil microbiology . Marcel Dekker, New York. - Arrebola E, Jacobs R, Korsten L, Iturin. 2010. A is the principal inhibitor in the biocontrol activity of
Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens.J. Appl. Microbiol. 108 : 386-395. - An Y, Kang S, Kim KD, Hwang BK, Jeun Y. 2010. Enhanced defense responses of tomato plants against late blight pathogen
Phytophthora infestans by pre-inoculation with rhizobacteria.Crop Prot. 29 : 1406-1412. - Júnior VL, Maffia LA, Romeiro RS, Mizubuti ESG. 2006. Biocontrol of tomato late blight with the combination of epiphytic antagonists and rhizobacteria.
Biol. Control. 38 : 331-340. - Akutsu K, Hirata A, Yamamoto M, Hirayae K, Okuyama S, Hibi T. 1993. Growth inhibition of
Botrytis spp. bySerratia marcescens B2 isolated from tomato phylloplane.Ann. Phytopathol. Soc. Jpn. 59 : 18-25. - Sutton JC, Peng G. 1993. Biocontrol of
Botrytis cinerea in strawberry leaves.Phytopathology 83 : 615-621. - Paul B. 1999. Suppression of
Botrytis cinerea causing the grey mould disease of grape-vine by an aggressive mycoparasite,Pythium radiosum .FEMS Microbiol. Lett. 176 : 25-30. - Harley JL, Waid JS. 1975. A method of studying active mycelia on living roots and other surfaces in the soils.
Trans. Br. Mycol. Soc. 38 : 104-118. - Bharathi R, Vivekananthan R, Harish S, Ramanathan A, Samiyappan R. 2004.
Rhizobacteria based bio-formulations for the management of fruit rot infection in chillies.Crop Prot. 23 : 835-843. - Sheng JX, Kim BS. 2014. Biocontrol of fusarium crown and root rot and promotion of growth of tomato by
Paenibacillus strains isolated from soil.Mycobiology 42 : 158-166. - Perneel M, Heyrman J, Adiobo A, De Maeyer K, Raaijmakers JM, De Vos P, Höfte M. 2007. Characterization of CMR5c and CMR12a, novel fluorescent
Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity.J. Appl. Microbiol. 103 : 1007-1020. - Trivedi P, Pandey A. 2007. Biological hardening of micropropagated
Picrorhiza kurrooa Royel ex Benth., an endangered species of medical importance.World J. Microbiol. Biotechnol. 23 : 877-878. - Roberts WK, Selitrennikoff CP. 1988. Plant and bacterial chitinases differ in antifungal activity.
J. Gen. Microbiol. 134 : 169-176. - Cappuccino JC, Sherman N. 2006. Microbiology: a laboratory manual, 6th ed. Pearson Education, Inc, San Francisco.
- Lorck H. 1948. Production of hydrocyanic acid by bacteria.
Physiol. Plant. 1 : 142-146. - Schwyn B, Neilands JB. 1987. Universal chemical assay for the detection and determination of siderophores.
Anal. Biochem. 160 : 47-56. - Bric JM, Bostock RM, Silverstone SE. 1991. Rapid
in situ assay for indole acetic acid production by bacteria immobilized on a nitrocellulose membrane.Appl. Environ. Microbiol. 57 : 535-538. - Goswami D, Vaghela H, Parmar S, Dhandhukia P, Thakker JN. 2013. Plant growth promoting potentials of
Pseudomonas spp. strain OG isolated from marine water.J. Plant Interact. 8 : 281-290. - Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 1991. 16S ribosomal DNA amplification for phylogenetic study.
J. Bacteriol. 173 : 697-703. - Reysenbach AL, Giver LJ, Wickham GS, Pace NR. 1992. Differential amplification of rRNA genes by polymerase chain reaction.
Appl. Environ. Microbiol. 58 : 3417-3418. - Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0.
Mol. Biol. Evol. 30 : 2725-27299. - Oh BJ, Kim KD, Kim YS. 1999. Effect of cuticular wax layers of green and red pepper fruits on infection by
Colletotrichum gloeosporioides .J. Phytopathol. 147 : 547-552. - Guo JH, Qi HY, Guo YH, Ge HL, Gong LY, Zhang LX. 2004. Biocontrol of tomato wilt by plant growth-promoting rhizobacteria.
Biol. Control 29 : 66-72. - Jeffries P, Gianinazzi S, Perotto S, Turnau K, Barea JM, IRRI. CROPSTAT for Windows, version 7.2.3. 2007; Metro Manila, Philippines. 2003. The contribution of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and soil fertility.
Biol. Fertil Soils 37 : 1-16. - Atkinson A, Watson CA. 2000. The beneficial rhizosphere: a dynamic entity.
Appl. Soil Ecol. 48 : 99-104. - Garbeva P, van Veen JA, van Elsas JD. 2004. Microbial diversity in soil: selection of microbial populations by plant and soil type and implications for disease suppressiveness.
Ann. Rev. Phytopathol. 42 : 243-270. - Paulitz TC, Zhou T, Rankin L. 1992. Selection of rhizosphere bacteria for biological control of
Pythium aphanidermatum on hydroponically grown cucumber.Biol. Control 2 : 226-237. - Bakthavatchalu V, Dey S, Xu Y, Noel T, Jungsuwadee P, Holley AK,
et al . 2011. Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Polγ against UV-induced inactivation.Oncogene 31 : 2129-2139. - Landa BB, Hervas A, Bethiol W, Jimenez-Diaz DR. 1997. Antagonistic activity of bacteria from the chickpea rhizosphere against
Fusarium oxysporum f. sp.ciceris .Phytoparasitica 25 : 305-318. - Saravanan T, Muthusami M, Marimuthu T. 2004. Effect of
Pseudomonas fluorescens on fusarium wilt pathogen in banana rhizosphere.J. Biol. Sci. 4 : 192-198. - Ashwini N, Srividya S. 2014. Potentiality of
Bacillus subtilis as biocontrol agent for management of anthracnose disease of chilli caused byColletotrichum gloeosporioides OGC1.3Biotech. 4 : 127-136. - Williams GE, Asher MJC. 1996. Selection of rhizobacteria for the control of
Pythium ultimum andAphanomyces cochlioides on sugarbeet seedlings.Crop Prot. 15 : 479-486. - Baker R. 1968. Mechanisms of biological control of soil-borne pathogens.
Annu. Rev. Phytopathol. 6 : 263-294. - Xu GW, Gross DC. 1986. Selection of fluorescent pseudomonads antagonistic to
Erwinia carotovora and suppressive of potato seed piece decay.Phytopathology 76 : 414-422. - Majeed A, Abbasi MK, Hameed S, Imran A, Rahim N. 2015. Isolation and characterization of plant growth-promoting rhizobacteria from wheat rhizosphere and their effect on plant growth promotion.
Front. Microbiol. 6 : 198. - Voisard C, Keel C, Haas D, Dèfago G. 1989. Cyanide production by
Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions.EMBO J. 8 : 351-358. - Bashan Y, De-Bashan LE. 2010. How the plant growth-promoting bacterium
Azospirillum promotes plant growth-a critical assessment.Adv. Agron. 108 : 77-136. - Muthamilan M, Jeyyarajan R. 1996. Integrated management of
Sclerotium root rot of groundnut involving ofTrichoderma harzianum ,Rhizobium and Carbendazim.Indian J. Mycol. Plant Pathol. 26 : 204-209. - van Peer R, Niemann GJ, Schippers B. 1991. Induced resistance and phytoalexin accumulation in biological control of fusarium wilt of carnation by
Pseudomonas sp. Strain WCS417r.PDF.Phytopathology 81 : 728-734. - Morrissey JP, Walsh UF, O'Donnell A, Moënne-Loccoz Y, O'Gara F. 2002. Exploitation of genetically modified inoculants for industrial ecology applications.
Antonie Van Leeuwenhoek 81 : 599-606.