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Suppression of Alternaria blight disease and plant growth promotion of mustard (Brassica juncea L.) by antagonistic rhizosphere bacteria
Applied Soil Ecology xxx (xxxx) xxx–xxx
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Suppression of Alternaria blight disease and plant growth promotion of mustard (Brassica juncea L.) by antagonistic rhizosphere bacteria R. Sharma, S. Sindhu, S.S. Sindhu
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Department of Microbiology, CCS Haryana Agricultural University, Hisar 125004, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Mustard Alternaria brassicae Rhizosphere bacteria Disease suppression Biological control Plant growth
Indian mustard (Brassica juncea L.) is an important oilseed crop. Alternaria brassicae causes blight disease on this crop leading to 10–70% yield losses. Rhizobacterial isolates were tested in this study for use as biological control agent for suppression of the disease. Three hundred and eighty-three rhizobacterial isolates were screened for the antagonistic activity and 20.88% isolates inhibited the growth of fungi Alternaria brassicae on modified LB medium plates. Six rhizobacterial isolates HMM44, HMM89, HMR25, HMR32, HMR33 and HMR70 showed significant antagonistic activity. Antagonistic rhizobacterial isolates were analyzed for their disease suppression and plant-growth promoting activities such as production of ALA, IAA, HCN and siderophore. Maximum IAA production was shown by isolate HMR48 and high ALA production was observed in isolates HMM21 and HMM49. Five isolates i.e. HMR25, HMR29, HMR52, HMR69 and HMR73 showed HCN production. Under pothouse conditions, inoculation of isolates HMR25, HMR48 and WHA64 with fungi caused 100, 80.0 and 80.0% disease control, respectively at 50 DAS (days after sowing). Inoculation of rhizobacterial isolates HMR70, HMR48 and HMR25 with A. Brassicae caused 83.33, 83.33 and 100.0% disease control, respectively at 75 days of plant growth. Rhizobacterial isolates HMR48 and HMR25 also showed stimulation of mustard growth. These rhizobacterial isolates could further be assessed for their disease control and plant growth promotion potential under field conditions for their subsequent use as biofertilizer and biocontrol agents.
1. Introduction
over the normal management practices (Hossain and Mian, 2005; Meena et al., 2011). Fungicide sprays are although effective in controlling the various fungal diseases but their extensive use is environmentally unsafe and also uneconomical. Other problems include development of resistant races of pathogens. Therefore, it is imperative to develop some alternate strategies to control plant diseases. Due to development of fungicide resistant strains in plant pathogens and environmental pollution problems, biocontrol agents have been characterized to substitute the recommended chemicals (Glick et al., 1999; Sindhu et al., 2009). Rhizosphere bacteria (rhizobacteria) were found to suppress the plant diseases by various mechanisms viz., production of antibiotics, production of hydrolytic enzymes (Sindhu and Dadarwal, 2001), hydrocyanic acid (Sarhan and Shehata, 2014), stimulation of phytoalexins or flavonoid-like compounds in roots (Goel et al., 2001) or by production of siderophores, which chelate metal cations rendering them unavailable for pathogenic forms (Sahu and Sindhu, 2011). Thus, biological control offers an alternative to use of costly agrochemicals by producing low cost environmental friendly control measures using antagonistic microorganisms that reduces the number and activity of plant
The major constraints in growing oilseed mustard crop are diseases, aphid pests, weeds and abiotic stresses including frost injury and salt, which are responsible for reduction in growth, yield and oil production. Alternaria blight disease, caused by Alternaria brassicae and A. brassicicola, is among the important diseases of Indian mustard which has been reported from all the continents of the world, causing 10–70% yield losses depending upon the crop speices (Kolte et al., 1987; Meena et al., 2011). Alternaria affects most cruciferous crops, including broccoli and cauliflower (Brassica oleracea L. var. botrytis L.), field mustard and turnip (B. rapa L. (synonym: B. campestris L.), Chinese mustard (B. juncea), Chinese or celery cabbage (B. pekinensis), cabbage (B. oleracea var. capitata), rape (B. campestris), and radish (Raphanus sativus) (Meena et al., 2016). A. brassicae and A. Brassicicola are cosmopolitan in their distribution. The plant diseases caused by fungi are mostly controlled by application of agrochemicals (fungicides and pesticides) and in some cases by cultural practices (Meena et al., 2016). Application of the fungicides mancozeb and iprodione (2 g L−1) recorded the best Alternaria blight reduction (93.2%) and increased the seed yield by 207.8%
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Corresponding author. E-mail address: [email protected] (S.S. Sindhu).
https://doi.org/10.1016/j.apsoil.2018.05.013 Received 12 January 2018; Received in revised form 15 May 2018; Accepted 19 May 2018 0929-1393/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Sharma, R., Applied Soil Ecology (2018), https://doi.org/10.1016/j.apsoil.2018.05.013
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cultures to inhibit fungal growth on modified LB media was assessed by the following formula: Halo zone to growth ratio = Halo zone area (0.5 H2)/Growth area (0.5 G2)
pathogens (Sindhu et al., 2016; Bach et al., 2016). These microbial populations in the rhizosphere, having the ability to act as biocontrol agents, may benefit the plant in a variety of ways, including: (i) increased recycling, solubilization and uptake of mineral nutrients (Pii et al., 2015), (ii) synthesis of vitamins, amino acids, auxins and gibberellins (Malik and Sindhu, 2011) and (iii) antagonism with potential plant pathogens (Sindhu et al., 2016). Certain PGPR strains were also found to protect the plants through mechanism associated with induced systemic resistance (ISR) against pathogens that cause foliar disease symptoms (Weller et al., 2012). Recently, seed bacterization with species of Azotobacter, Rhizobium, Bacillus and Pseudomonas were found to inhibit the growth of soil-borne root infecting fungi and suppressed the various fungal diseases resulting in enhanced plant growth as well as yield of various crops under green house and field conditions (Siddiqui et al., 2001; Sindhu et al., 2016). Rahman et al. (2016) characterized effective Bacillus amyloliquefaciens subsp. Plantarum from rhizospheric soil samples as potential bacterial biocontrol agents against white mold disease caused by Sclerotinia sclerotiorum. The production of hydrolytic enzymes and the plant growth-promotional attributes of these isolates confirmed their multifaceted potential. In this study, rhizobacterial isolates were tested for growth inhibition of the fungi Alternaria brassicae and for their inoculation effect on growth of mustard plants.
2.4. Production of indole acetic acid (IAA) and δ-aminolevulinic acid (ALA) For estimation of IAA production, different bacterial cultures were inoculated in duplicate in 30 ml LB broth supplemented with L-tryptophan @ 100 μg ml−1. Inoculated broth flasks were incubated at 28 ± 2 °C for 48 h under stationary conditions of growth. After centrifugation of culture samples at 10,000 rpm for 15 min (Remi Instruments, Mumbai India), IAA was determined in the supernatant by the method as described by Malik and Sindhu (2011). For ALA determination, LB broth (10 ml) supplemented with 15 mM glycine and succinate, was inoculated with different rhizobacterial cultures(in duplicate) and incubated at 28 ± 2 °C for 48 h. After centrifugation of culture samples at 10,000 rpm for 15 min, ALA was determined in the supernatant using Mauzerall and Granick (1955) method. 2.5. Qualitative detection of siderophore and hydrogen cyanide production
2. Materials and methods
The chrome azurol sulfonate (CAS) assay was used for detection of siderophore production (Schwyn and Neilands, 1987). Cultures were spot inoculated onto the blue agar plates and incubated at 28 ± 2 °C for 48 h. The size of yellow orange halos around the bacterial colony indicated siderophore production by the rhizobacterial cultures. For determination of hydrogen cyanide production by bacterial isolates, cultures were grown on King’s B medium broth supplemented with 4.4 g L−1 glycine (Alstrom and Burns, 1989). Three millimetre strips of Whatman No. 42 filter paper were sterilized and soaked in 0.5% picric acid and 2% sodium carbonate solution. These strips were hanged alongside the cotton plug in the test tube inoculated with different bacterial cultures. After incubation at 28 ± 2 °C for 5 days, color of the filter paper strips was changed from yellow to orange red due to production of hydrogen cyanide by rhizobacterial cultures.
2.1. Isolation of bacteria from rhizosphere soil Rhizosphere soil samples were collected from different fields of mustard grown in CCS Haryana Agricultural University, Hisar farm (29°09′ 51.66″N; 75°41′2.94″E) at 45 and 60 days of plant growth. The mustard crop was grown in the sandy loam soil having the characteristics i.e., organic carbon, 0.60%; pH, 7.5; electrical conductivity, 0.14 ds m−1, available phosphorus, 17 kg ha−1, available K2O, 617 kg ha−1. Composite rhizosphere soil samples were diluted up to 10−4 and plated on Luria Bertani (LB) medium plates. Based on morphological and pigment production characteristics, different bacterial colonies were selected after 3 days of incubation at 28 ± 2 °C. Isolated colonies of bacteria were purified by streak plate method and transferred on LB agar medium slopes.
2.6. Coinoculation effect of rhizobacterial isolates with Alternaria brassicae on mustard
2.2. Isolation of pathogenic fungi Alternaria brassicae from diseased mustard plant
Five rhizobacterial isolates i.e., HMR25, HMR48, HMR70, JMM16 and WHA64 were used for coinoculation (Sindhu et al., 2002). The earthen pots (10 kg capacity) were filled with sandy loam soil and river sand mixed in 70:30 ratios. Growth suspension of rhizobacterial isolates grown for 48 h on LB medium slopes was made in 5 ml of sterilized water. Seeds of mustard var. RH749 were inoculated with 5 ml bacterial growth suspension for 1 h. The viable count in the broth was kept 108–109 cells ml−1. Growth of 5 days-old A. Brassicae was harvested from PDA plates and fungal growth suspension was prepared in sterilized saline water. Fungal growth suspension (100 ml) was mixed in the 10 kg soil: sand mixture in earthen pots in coinoculation treatments. Uninoculated seeds were sown as control. The plants were grown in the pot house under day light conditions. Plants were watered as and when required. The plants were uprooted at 50 and 75 days of plant growth and observations were taken for plant dry weight and disease index. After washing with tap water, roots were dried in the folds of filter paper. Shoot portions of the plants were dried in oven at 90 °C for 24 h and weighted. On the basis of symptoms observed, percent disease index and percent disease control were calculated by following equation:
Diseased mustard leaves with concentric lesions on the leaf surface were collected from University farm (29°08′ 22.11″N; 75°42′ 16.92″E). Lesions were cut and sterilized with 0.1% HgCl2. The sterilant was removed through successive treatment with distilled water. Then, lesion spots were inoculated on potato dextrose agar (PDA) plates and incubated at 25 ± 2 °C in BOD incubator for 5–7 days. After sporulation, the morphological characteristics of spores were checked microscopically. The fungal cultures were stored at 4 °C in refrigerator for further use. 2.3. Screening of rhizobacterial isolates for antagonistic activity The antagonistic interaction of rhizobacterial isolates with Alternaria brassicae was studied by the spot test method (Sindhu et al., 1999) on modified LB medium plates. The fungus A. Brassicae was grown on PDA slants and spore suspension of the fungi was prepared in 3 ml sterilized water. Fungal spore suspension (200 µl) was spread over modified LB medium plates. Plates were incubated for 2 h in BOD incubator at 28 ± 2 °C. Bacterial growth suspension (2.0 μl) prepared from 48-h old growth of the rhizobacterial isolates was spotted on preseeded plates. Growth inhibition of fungi was recorded after 3 days of incubation at 28 + 2 °C. Antagonistic activity of the rhizobacterial
%Disease incidence(DI) =
2
No. of diseased plants × 100 Total no. of plants
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%Disease control No.of infected plants in control−No.of infected plants in treatment = No of infected plants in control
2.7. Statistical analysis Completely Randomized Design (CRD) was used for experimental data analysis. All determinations were carried out in triplicate and data represented are average values of three replications. Standard Error of Means ± (SEM) values were calculated to determine the significant differences between treatment means. The C.D. and C.V. values represent coefficient of deviation and coefficient of variation, respectively.
Fig. 1. Frequency of antagonistic isolates for fungal growth inhibition, Halo zone to growth ratio has been reported for 80 different rhizobacterial isolates.
(Table 2). More ALA production was observed in isolates HMR57 (19.95 µg ml−1) and HMR32 (16.33 µg ml−1). Out of 23 selected bacterial isolates tested, only six isolates HMR25, HMR29, HMR52, HMR60, HMR69 and HMR73 showed HCN production. Eight rhizobacterial isolates i.e. HMM49, HMM97, HMR25, HMR32, HMR52 HMR56, HMR69, and JMM16 showed siderophore production.
3. Results 3.1. Screening of bacterial cultures for growth inhibition of fungi All the 383 bacterial isolates were screened for their antagonistic interaction against fungi Alternaria brassicae on modified LB medium plates. The fungal growth inhibition zone varied with different bacterial isolates. Only, 80 isolates inhibited the growth of A. brassicae. Rhizobacterial isolates HMM51, HMR25, HMR33 and HMR70 showed fungal inhibition ratio of more than 4.0 (halozone to growth ratio) (Table 1, Figs. 1 and 2a, b). Thirty-one isolates showed > 3.0 halozone to growth ratio whereas thirteen isolates showed > 2.0 halozone to growth ratio of fungal inhibition. Thirty-two isolates comparatively showed less inhibition of the fungus and the ratio of halozone to growth varied between 1.0 and 2.0. Overall, only 20.88% isolates showed antagonistic activity against A. brassicae and rest did not inhibit the growth of fungi.
3.3. Coinoculation effect of Alternaria brassicae and rhizobacterial isolates for disease control and plant growth On the basis of variation in antagonistic activity and beneficial attributes, five rhizobacterial isolates i.e., HMR25, HMR48, HMR70, JMM16 and WHA64 were selected for inoculation effect on mustard plants seeds under pot house conditions. At 50 days of growth, coinoculation of fungi with rhizobacterial isolates JMM16 and HMR48 showed 140.0 and 96.9% increase in shoot dry weight as compared to uninoculated control, respectively (Table 3, Fig. 3). Inoculation with rhizobacterial isolates HMR70 and JMM16 both caused 107.1% increase in root dry weight as compared to uninoculated control. Disease control was 100% in HMR25 inoculated plants (Table 3). Rhizobacterial isolates HMR48 and WHA64 both showed 80.0% disease control in comparison to uninoculated control. At 75 days of growth, coinoculation of bacterial isolates HMR70, HMR48 and HMR25 with fungi showed 94.0, 77.5 and 95.5% increase in shoot dry weight as compared to control, respectively (Table 3, Fig. 3). Coinoculation of fungi and rhizobacterial isolates HMR70 and HMR48 showed only 14.8 and 17.5% increase in root dry weight in comparison to uninoculated control, respectively. Disease control was 100% in HMR25 inoculated plants (Table 3, Fig. 4) and inoculation of rhizobacterial isolates HMR70 and HMR48 both caused 83.33% disease control. It was observed that rhizobacterial isolates HMR70 showed significant increase in shoot dry weight at both the stages of plant growth (Fig. 5), and inoculation of isolates JMM16 and HMR48 caused significant gains in shoot dry weight. Coinoculation of all the rhizobacterial isolates with A. brassicae improved the shoot dry weight at 50 days of plant growth. However, only four rhizobacterial isolates i.e., HMR25, HMR48, HMR70 and JMM16 showed significant gains in plant dry weight at 75 days of plant growth. Inoculation with rhizobacterial isolate HMR25 caused 100% disease control at both the stages of plant growth, whereas inoculation with rhizobacterial isolates HMR48 andHMR70 showed 83.33% disease control at 75 days of growth as compared with respective control (Figs. 3 and 4). Thus, inoculation of rhizobacterial isolates HMR70 and JMM16 enhanced the plant dry weight of mustard as compared to uninoculated control plants at both 50 and 75 days of growth (Table 3, Figs. 3–5). In case of inoculation of fungi and bacteria, isolates HMR70, HMR48 and HMR25 showed significant increase in shoot dry weight in comparison to control. The inoculation of bacterial isolate HMR70 and fungi, increased shoot dry weight by 81.5 and 94.0% at 50 and 75 DAS, respectively. Whereas, inoculation of bacterial isolate HMR25 and fungi increased the shoot dry weight by 72.3 and 95.5% at 50 and 75 DAS, respectively. At 50 DAS, isolates HMR70, JMM16, HMR48, WHA64 and HMR25 showed 60.0, 40.0, 80.0, 80.0 and 100% disease control,
3.2. Growth promoting characteristics of rhizobacterial isolates Twenty-three selected antagonistic rhizobacterial isolates were analyzed for their plant-growth promoting activities like production of ALA, IAA, HCN and siderophore. Maximum IAA production (12.11 µg ml−1) was shown by isolate HMR48 followed by JMM16 (2.17 µg ml−1) and12 antagonistic isolates did not produce IAA Table 1 Halo zone to growth ratio of different rhizobacterial isolates. Rhizobacterial Isolates
Inhibition of A. brassicae (Halo zone to growth ratio)*
CP109 HMM21 HMM49 HMM97 HMR3 HMR25 HMR29 HMR32 HMR33 HMR39 HMR48 HMR52 HMR55 HMR56 HMR57 HMR59 HMR60 HMR69 HMR70 HMR73 JMM16 WHA64 WHA99
1.45 1.22 3.43 2.44 2.86 5.0 3.24 3.83 4.68 3.44 1.32 1.54 1.32 2.54 2.11 1.65 1.25 1.23 4.12 3.22 2.51 1.58 3.15
* Halo zone to growth ratio = Halo zone area (0.5 H2) Growth area (0.5 G2). 3
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Fig. 2. (a, b) Growth inhibition of A. brassicae by different rhizobacterial isolates. Table 2 Beneficial attributes of different rhizobacterial isolates. Rhizobacterial isolates
IAA (µg/ml)
ALA (µg/ml)
HCN productionδ
Siderophore productionα
CP109 HMM21 HMM49 HMM97 HMR3 HMR25 HMR29 HMR32 HMR33 HMR39 HMR48 HMR52 HMR55 HMR56 HMR57 HMR59 HMR60 HMR69 HMR70 HMR73 JMM16 WHA64 WHA99
0.99 0.24 1.65 0.96 0 0.36 1.77 0.09 0 0 12.11 2.68 1.80 0.33 0 1.02 0 0 1.47 1.02 2.17 1.20 1.17
8.12 14.74 14.45 13.53 9.66 8.74 14.66 16.33 10.29 17.99 10.53 10.37 10.12 11.58 19.95 12.03 0 10.37 8.66 10.74 12.87 10.91 11.58
− − − − − + 2+ − − − − 2+ − − − − + 2+ − 2+ − − −
− − + 2+ − 2+ − + − − − ± − 2+ − − − 2+ − − + − −
Table 3 Effect of single and mixed inoculation on mustard growth under pot-house conditions. Treatments
DAS
Root dry weight (mg/ plant)
Shoot dry weight (mg/ plant)
Disease incidence (%)
Disease control (%)
Soil
50 75 50 75 50 75 50 75 50 75
20 ± 1.00 42 ± 1.52 28 ± 2.51 68 ± 1.15 46 ± 1.52 74 ± 1.52 58 ± 1.52 108 ± 1.15 42 ± 1.15 85 ± 1.52
49 ± 1.52 78 ± 2.51 72 ± 1.52 178 ± 1.52 65 ± 0.57 134 ± 1.52 170 ± 1.15 382 ± 1.15 118 ± 1.00 260 ± 3.05
– – – – 71.42 85.71 – – 28.57 14.28
– – – – – – – – 60.0 83.33
50 75 50 75
58 88 45 72
188 276 156 235
± ± ± ±
1.52 1.52 1.52 0.57
– – 42.85 42.85
– – 40.0 50.0
50 75 50 75
49 ± 1.52 104 ± 1.52 41 ± 0.57 87 ± 1.00
152 316 128 258
± ± ± ±
1.15 2.64 2.51 1.52
– – 14.28 14.28
– – 80.0 80.0
50 75 50 75
47 70 32 58
± ± ± ±
1.00 1.15 1.52 1.15
132 252 119 194
± ± ± ±
1.15 0.57 1.15 1.15
– – 14.28 28.57
– – 80.0 66.66
50 75 50 75
40 82 34 75
± ± ± ±
1.52 1.00 1.00 0.57
126 271 112 262
± ± ± ±
1.00 1.00 1.00 1.15
– – 0 0
– – 100.0 100.0
50 75 50 75
4.080 3.68 5.821 2.80
– – – –
– – – –
Soil + RDF T2 + A. brassicae T2 + HMR70 T2 + A. brassicae + HMR70 T2 + JMM16 T2 + A. brassicae + JMM16 T2 + HMR48 T2 + A. brassicae + HMR48 T2 + WHA64
δ
On the basis of yellow orange halos after 2–5 days of incubation at 28 ± 2 °C, the production of siderophore by the rhizobacterial isolates was scored as: −: No production, +: Moderate production, 2+: high production. α The change in the color of the strips from yellow to orange red was observed for hydrogen cyanide production, by the method of Alstrom and Burns (1989). On the basis of change in the color of the strips from yellow to orange red after 2–5 days of incubation at 28 ± 2 °C, the production of HCN by rhizobacterial isolates was scored as: −: No production, +: Moderate production, ++: high HCN production.
T2 + A. brassicae + WHA64 T2 + HMR25 T2 + A. brassicae + HMR25 CD CV
respectively. At 75 DAS, rhizobacterial isolates HMR70, JMM16, HMR48, WHA64 and HMR25 showed 83.33, 50.0, 83.33, 66.66 and 100% disease control, respectively (Table 3).
± ± ± ±
0.57 1.15 1.52 1.52
3.91 5.04 1.90 1.25
Values given are average value of three plants. Disease incidence is the% of plants infected and disease control is the% reduction of diseased plants after inoculation with bacteria. The values of nodule fresh and shoot dry weight are calculated as per plant basis. C.D. and C.V. values represent coefficient of deviation and coefficient of variation, respectively. SEM (Standard Error of Means) values are represented as ( ± ).
4. Discussion Antagonistic microorganisms play a major role in microbial equilibrium and serve as powerful agents for biological disease control (Ana et al., 2009). The applications of biocontrol agents in the protection of some commercially important crops are increasingly capturing the imagination of many plant pathologists and microbiologists. In this study, 383 rhizobacterial isolates were studied for growth inhibition of A. brassicae on LB medium plates. It was found that only 20.88% isolates possess the ability to inhibit the pathogenic fungi A. brassicae in cultural conditions (Table 1, Figs. 1 and 2a, b).Foroutan et al. (2005)
obtained 132 isolates representing Pseudomonas and Bacillus species from the rhizosphere of wheat and thirty-two isolates showed antagonism against Fusarium graminearum. Bacillus megaterium isolated from tea rhizosphere produced siderophore and showed reduction in disease intensity (Chakraborty et al., 2010). To know the nature of antagonistic compounds produced by rhizobacterial isolates, HCN and siderophore production was examined in 4
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the selected rhizobacterial isolates. Isolate HMR25 showed siderophore production and also produced HCN (Table 2). Isolate JMM16 produced siderophore only. In similar studies, rhizosphere competent Mesorhizobium loti MP6 was found to produce hydrocyanic acid under normal growth conditions and enhanced the growth of Indian mustard (Brassica campestris) (Chandra et al., 2007). Jošić et al. (2015) reported that PGP isolates, Q16 and B25, showed the best antifungal activity against Trichoderma viride and good antifungal effect against Aspergillus fumigates and Aspergillus niger, while Penicillium verrucosum was the most resistant fungus. Three bacterial isolates HMR32, HMR39 and HMR57 showed more than 15 μg ml−1 ALA production (Table 2). Bacterial isolate HMR48 produced significant amount of IAA and ALA, and also inhibited the growth of A. brassicae. On the other hand, isolate HMR25 having highest antagonistic activity produced only ALA. Loper and Schroth (1986) observed a significant linear relationship between IAA accumulation of the rhizobacterial strains and decreased root elongation of sugar beet seedlings. Similar concentration dependent effects of IAA on stimulation or inhibition of root/shoot growth has been reported in earlier studies (Arshad and Frankenberger, 1991). Yaish et al. (2015) isolated bacterial strains that produced the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and the plant growth regulatory hormone indole-3-acetic acid. Some strains also chelated ferric iron (Fe3+), solubilized potassium, phosphorus and zinc, and also produced ammonia. Sarhan and Shehata (2014) tested four strains of plant growth promoting rhizobacteria (Bacillus subtilis, Paenibacillus polymyxa, Pseudomonas fluorescens and Pseudomonas putida) and Sinorhizobium meliloti for their antibiosis toward damping-off disease and yield of alfalfa crop. In vitro, the four PGPR strains produced hydrogen cyanide, indole-3-acetic acid, siderophore, solubilized insoluble phosphate and showed protease and β-1, 3-glucanaseactivities, whereas S. meliloti produced IAA and solubilized insoluble phosphate only. Such multi-trait rhizobacterial isolates having fungal growth inhibiting capability and plant growth promoting ability could have better potential for use as efficient biofertilizers and biopesticides under field conditions. In similar studies, various PGP bacteria were evaluated for plant
Shoot dry weight (mg/plant)
400 Control
350
HMR70
300
JMM16
250
HMR48
200
WHA64
150
HMR25
100 50 0 50
75 Days after sowing
Fig. 3. Coinoculation effect of rhizobacteria and fungi on shoot dry weight of mustard plants at 50 and 75 days of growth.
Fig. 4. Disease incidence (%) in case of different rhizobacterial isolates (DAS: Days after sowing).
Fig. 5. Growth of mustard plants inoculated with rhizobacterial isolates and/or fungi at 50 and 75 days. T2: Control + RDF; T3: T2 + A. brassicae. T4: T2 + Isolate HMR70; T5: T2 + HMR70 + A. brassicae. T6: T2 + Isolate JMM16; T7: T2 + JMM16 + A. brassicae. 5
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growth promoting (PGP) characteristics, biological control and rhizosphere competence (Bach et al., 2016). All bacteria showed competitive characteristics that could enhance rhizosphere competence either through hydrolytic enzyme production or through antagonistic activities. Both B. mycoides B38V and B. cepacia 89 showed remarkable antifungal activity in addition to other PGP characteristics, representing potential inoculants and biocontrol agents. Ayuke et al. (2017) studied the biological control of plant diseases through the addition of microbial biocontrol agents along with the earthworms as an environmentally friendly alternative to the chemical control of plant diseases. Beneficial bacterium Bacillus amyloliquefaciens and the earthworms Aporrectodea caliginosa or Aporrectodea longa were found to reduce the disease in oilseed rape (Brassica napus), when challenged with the pathogen Alternaria brassicae.
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Rahman, M.M., Hossain, D.M., Suzuki, K., Shiiya, A., Suzuki, K., Dey, T.K., Nonaka, M., Harada, N., 2016. Suppressive effects of Bacillus spp. on mycelia, apothecia and sclerotia formation of Sclerotinia sclerotiorum and potential as biological control of white mold on mustard. Aust. Plant Pathol. 45, 103–117. Sahu, G.K., Sindhu, S.S., 2011. Disease control and plant growth promotion of green gram by siderophore producing Pseudomonas sp. Res. J. Microbiol. 6 (10), 735–749. Sarhan, E.A.D., Shehata, H.S., 2014. Potential plant growth-promoting activity of Pseudomonas spp. and Bacillus spp. as biocontrol agents against damping off in alfalfa. Plant Pathol. J. 13 (1), 48–52. Schloter, M., Nannipieri, P., Sørensen, S.J., van Elsas, J.D., 2018. Microbial indicators for soil quality. Biol Fertil Soils 54, 1–10. http://dx.doi.org/10.1007/s00374-0171248-3. Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection and determination of siderophore. Anal. Biochem. 160, 47–56. Siddiqui, I.A., Ehteshamul-Haque, S., Shaukat, S.S., 2001. Use of rhizobacteria in the control of root-rot knot disease complex of mungbean. J. Phytopathol. 149, 337–346. Sindhu, S.S., Dadarwal, K.R., 2001. Chitinolytic and cellulolytic Pseudomonas sp. antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp. Cicer in chickpea. Microbiol. Res. 156 (4), 353–358. Sindhu, S.S., Suneja, S., Goel, A.K., Parmar, N., Dadarwal, K.R., 2002. Plant growth promoting effects of Pseudomonas sp. on coinoculation with Mesorhizobium sp. Cicer strain under sterile and “wilt sick” soil conditions. Appl. Soil Ecol. 19 (1), 57–64. Sindhu, S.S., Rakshiya, Y.S., Sahu, G., 2009. Biological control of soil borne plant pathogens with rhizosphere bacteria. Pest Technol. 3, 10–21. Sindhu, S.S., Gupta, S.K., Dadarwal, K.R., 1999. Antagonistic effect of Pseudomonas spp. on pathogenic fungi and enhancement of plant growth in green gram (Vigna radiata). Biol. Fertil. Soils 29, 62–68. Sindhu, S.S., Sehrawat, A., Sharma, R., Dahiya, A., 2016. Biopesticides: use of rhizosphere bacteria for biological control of plant pathogens. Defence Life Sci. J. 1, 135–148. Weller, D.M., Mavrodi, D.V., van Pelt, J.A., 2012. Induced systemic resistance in Arabidopsis thaliana against Pseudomonas syringaepv. tomato by 2, 4-diacetyl phloroglucinol – producing Pseudomonas fluorescens. Phytopathology 102, 403–412. Yaish, M.W., Antony, I., Glick, B.R., 2015. Isolation and characterization of endophytic plant growth-promoting bacteria from date palm tree (Phoenix dactylifera L.) and their potential role in salinity tolerance. Antonie Van Leeuwen 107 (6), 1519–1532.
5. Conclusion Eco-friendly agricultural system has emerged as an important thrust area globally for long-term soil environmental sustainability and to minimize the environmental pollution associated with indiscriminate use of chemical pesticides and fertilizers. Rhizosphere microorganisms are currently being explored for their possible use as biocontrol agents in the integrated pest management programmes as an environmentfriendly alternative intervention. Moreover, the capacity of living soil to sustain biological productivity using beneficial microorganisms has been improved recently by using various methodological developments in the last decades leading to promotion of plant health while maintaining environmental quality (Schloter et al., 2018). Keeping in view, the problem of environmental pollution and health hazards, microorganisms inhabiting the mustard rhizosphere have been identified in this study for the biological control of the Alternaria blight disease. Rhizobacterial isolates were found to inhibit the growth of Alternaria brassicae on LB medium plates. The inoculation of the selected rhizobacterial isolates i.e., HMR25 and HMR48 on mustard caused 55–114.6% increase in shoot dry weight and 80–100% disease control at 50 and 75 days of plant growth under pot house conditions. These results offer new opportunities to estimate the contribution of the rhizosphere bacteria under field conditions in the biological control of Alternaria blight along with promotion of mustard plant growth for sustainable agriculture. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsoil.2018.05.013. References Alstrom, S., Burns, R.G., 1989. Cyanide production by rhizobacteria as a possible mechanism of plant growth inhibition. Biol. Fertil. Soils 7, 232–238. Anal, E.P., Moreno, V.M., Cordo, C., 2009. Biological control of Septoria tritici blotch on wheat by Trichoderma sp. under field conditions in Argentina. Biocontrol 54, 113–122. Arshad, M., Frankenberger Jr, W.T., 1991. Microbial production of plant hormones. Plant Soil 133, 1–8. Ayuke, F.O., Lagerlöf, J., Jorge, G., Söderlund, S., Muturi, J.J., Sarosh, B.R., Meijer, J., 2017. Effects of biocontrol bacteria and earthworms on the severity of Alternaria brassicae disease and the growth of oilseed rape plants (Brassica napus). Appl. Soil Ecol. 117, 63–69. Bach, E., dos Santos Seger, G.D., de Carvalho Fernandes, G., 2016. Evaluation of
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Short communication
Suppression of Alternaria blight disease and plant growth promotion of mustard (Brassica juncea L.) by antagonistic rhizosphere bacteria R. Sharma, S. Sindhu, S.S. Sindhu
⁎
Department of Microbiology, CCS Haryana Agricultural University, Hisar 125004, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Mustard Alternaria brassicae Rhizosphere bacteria Disease suppression Biological control Plant growth
Indian mustard (Brassica juncea L.) is an important oilseed crop. Alternaria brassicae causes blight disease on this crop leading to 10–70% yield losses. Rhizobacterial isolates were tested in this study for use as biological control agent for suppression of the disease. Three hundred and eighty-three rhizobacterial isolates were screened for the antagonistic activity and 20.88% isolates inhibited the growth of fungi Alternaria brassicae on modified LB medium plates. Six rhizobacterial isolates HMM44, HMM89, HMR25, HMR32, HMR33 and HMR70 showed significant antagonistic activity. Antagonistic rhizobacterial isolates were analyzed for their disease suppression and plant-growth promoting activities such as production of ALA, IAA, HCN and siderophore. Maximum IAA production was shown by isolate HMR48 and high ALA production was observed in isolates HMM21 and HMM49. Five isolates i.e. HMR25, HMR29, HMR52, HMR69 and HMR73 showed HCN production. Under pothouse conditions, inoculation of isolates HMR25, HMR48 and WHA64 with fungi caused 100, 80.0 and 80.0% disease control, respectively at 50 DAS (days after sowing). Inoculation of rhizobacterial isolates HMR70, HMR48 and HMR25 with A. Brassicae caused 83.33, 83.33 and 100.0% disease control, respectively at 75 days of plant growth. Rhizobacterial isolates HMR48 and HMR25 also showed stimulation of mustard growth. These rhizobacterial isolates could further be assessed for their disease control and plant growth promotion potential under field conditions for their subsequent use as biofertilizer and biocontrol agents.
1. Introduction
over the normal management practices (Hossain and Mian, 2005; Meena et al., 2011). Fungicide sprays are although effective in controlling the various fungal diseases but their extensive use is environmentally unsafe and also uneconomical. Other problems include development of resistant races of pathogens. Therefore, it is imperative to develop some alternate strategies to control plant diseases. Due to development of fungicide resistant strains in plant pathogens and environmental pollution problems, biocontrol agents have been characterized to substitute the recommended chemicals (Glick et al., 1999; Sindhu et al., 2009). Rhizosphere bacteria (rhizobacteria) were found to suppress the plant diseases by various mechanisms viz., production of antibiotics, production of hydrolytic enzymes (Sindhu and Dadarwal, 2001), hydrocyanic acid (Sarhan and Shehata, 2014), stimulation of phytoalexins or flavonoid-like compounds in roots (Goel et al., 2001) or by production of siderophores, which chelate metal cations rendering them unavailable for pathogenic forms (Sahu and Sindhu, 2011). Thus, biological control offers an alternative to use of costly agrochemicals by producing low cost environmental friendly control measures using antagonistic microorganisms that reduces the number and activity of plant
The major constraints in growing oilseed mustard crop are diseases, aphid pests, weeds and abiotic stresses including frost injury and salt, which are responsible for reduction in growth, yield and oil production. Alternaria blight disease, caused by Alternaria brassicae and A. brassicicola, is among the important diseases of Indian mustard which has been reported from all the continents of the world, causing 10–70% yield losses depending upon the crop speices (Kolte et al., 1987; Meena et al., 2011). Alternaria affects most cruciferous crops, including broccoli and cauliflower (Brassica oleracea L. var. botrytis L.), field mustard and turnip (B. rapa L. (synonym: B. campestris L.), Chinese mustard (B. juncea), Chinese or celery cabbage (B. pekinensis), cabbage (B. oleracea var. capitata), rape (B. campestris), and radish (Raphanus sativus) (Meena et al., 2016). A. brassicae and A. Brassicicola are cosmopolitan in their distribution. The plant diseases caused by fungi are mostly controlled by application of agrochemicals (fungicides and pesticides) and in some cases by cultural practices (Meena et al., 2016). Application of the fungicides mancozeb and iprodione (2 g L−1) recorded the best Alternaria blight reduction (93.2%) and increased the seed yield by 207.8%
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Corresponding author. E-mail address: [email protected] (S.S. Sindhu).
https://doi.org/10.1016/j.apsoil.2018.05.013 Received 12 January 2018; Received in revised form 15 May 2018; Accepted 19 May 2018 0929-1393/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Sharma, R., Applied Soil Ecology (2018), https://doi.org/10.1016/j.apsoil.2018.05.013
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cultures to inhibit fungal growth on modified LB media was assessed by the following formula: Halo zone to growth ratio = Halo zone area (0.5 H2)/Growth area (0.5 G2)
pathogens (Sindhu et al., 2016; Bach et al., 2016). These microbial populations in the rhizosphere, having the ability to act as biocontrol agents, may benefit the plant in a variety of ways, including: (i) increased recycling, solubilization and uptake of mineral nutrients (Pii et al., 2015), (ii) synthesis of vitamins, amino acids, auxins and gibberellins (Malik and Sindhu, 2011) and (iii) antagonism with potential plant pathogens (Sindhu et al., 2016). Certain PGPR strains were also found to protect the plants through mechanism associated with induced systemic resistance (ISR) against pathogens that cause foliar disease symptoms (Weller et al., 2012). Recently, seed bacterization with species of Azotobacter, Rhizobium, Bacillus and Pseudomonas were found to inhibit the growth of soil-borne root infecting fungi and suppressed the various fungal diseases resulting in enhanced plant growth as well as yield of various crops under green house and field conditions (Siddiqui et al., 2001; Sindhu et al., 2016). Rahman et al. (2016) characterized effective Bacillus amyloliquefaciens subsp. Plantarum from rhizospheric soil samples as potential bacterial biocontrol agents against white mold disease caused by Sclerotinia sclerotiorum. The production of hydrolytic enzymes and the plant growth-promotional attributes of these isolates confirmed their multifaceted potential. In this study, rhizobacterial isolates were tested for growth inhibition of the fungi Alternaria brassicae and for their inoculation effect on growth of mustard plants.
2.4. Production of indole acetic acid (IAA) and δ-aminolevulinic acid (ALA) For estimation of IAA production, different bacterial cultures were inoculated in duplicate in 30 ml LB broth supplemented with L-tryptophan @ 100 μg ml−1. Inoculated broth flasks were incubated at 28 ± 2 °C for 48 h under stationary conditions of growth. After centrifugation of culture samples at 10,000 rpm for 15 min (Remi Instruments, Mumbai India), IAA was determined in the supernatant by the method as described by Malik and Sindhu (2011). For ALA determination, LB broth (10 ml) supplemented with 15 mM glycine and succinate, was inoculated with different rhizobacterial cultures(in duplicate) and incubated at 28 ± 2 °C for 48 h. After centrifugation of culture samples at 10,000 rpm for 15 min, ALA was determined in the supernatant using Mauzerall and Granick (1955) method. 2.5. Qualitative detection of siderophore and hydrogen cyanide production
2. Materials and methods
The chrome azurol sulfonate (CAS) assay was used for detection of siderophore production (Schwyn and Neilands, 1987). Cultures were spot inoculated onto the blue agar plates and incubated at 28 ± 2 °C for 48 h. The size of yellow orange halos around the bacterial colony indicated siderophore production by the rhizobacterial cultures. For determination of hydrogen cyanide production by bacterial isolates, cultures were grown on King’s B medium broth supplemented with 4.4 g L−1 glycine (Alstrom and Burns, 1989). Three millimetre strips of Whatman No. 42 filter paper were sterilized and soaked in 0.5% picric acid and 2% sodium carbonate solution. These strips were hanged alongside the cotton plug in the test tube inoculated with different bacterial cultures. After incubation at 28 ± 2 °C for 5 days, color of the filter paper strips was changed from yellow to orange red due to production of hydrogen cyanide by rhizobacterial cultures.
2.1. Isolation of bacteria from rhizosphere soil Rhizosphere soil samples were collected from different fields of mustard grown in CCS Haryana Agricultural University, Hisar farm (29°09′ 51.66″N; 75°41′2.94″E) at 45 and 60 days of plant growth. The mustard crop was grown in the sandy loam soil having the characteristics i.e., organic carbon, 0.60%; pH, 7.5; electrical conductivity, 0.14 ds m−1, available phosphorus, 17 kg ha−1, available K2O, 617 kg ha−1. Composite rhizosphere soil samples were diluted up to 10−4 and plated on Luria Bertani (LB) medium plates. Based on morphological and pigment production characteristics, different bacterial colonies were selected after 3 days of incubation at 28 ± 2 °C. Isolated colonies of bacteria were purified by streak plate method and transferred on LB agar medium slopes.
2.6. Coinoculation effect of rhizobacterial isolates with Alternaria brassicae on mustard
2.2. Isolation of pathogenic fungi Alternaria brassicae from diseased mustard plant
Five rhizobacterial isolates i.e., HMR25, HMR48, HMR70, JMM16 and WHA64 were used for coinoculation (Sindhu et al., 2002). The earthen pots (10 kg capacity) were filled with sandy loam soil and river sand mixed in 70:30 ratios. Growth suspension of rhizobacterial isolates grown for 48 h on LB medium slopes was made in 5 ml of sterilized water. Seeds of mustard var. RH749 were inoculated with 5 ml bacterial growth suspension for 1 h. The viable count in the broth was kept 108–109 cells ml−1. Growth of 5 days-old A. Brassicae was harvested from PDA plates and fungal growth suspension was prepared in sterilized saline water. Fungal growth suspension (100 ml) was mixed in the 10 kg soil: sand mixture in earthen pots in coinoculation treatments. Uninoculated seeds were sown as control. The plants were grown in the pot house under day light conditions. Plants were watered as and when required. The plants were uprooted at 50 and 75 days of plant growth and observations were taken for plant dry weight and disease index. After washing with tap water, roots were dried in the folds of filter paper. Shoot portions of the plants were dried in oven at 90 °C for 24 h and weighted. On the basis of symptoms observed, percent disease index and percent disease control were calculated by following equation:
Diseased mustard leaves with concentric lesions on the leaf surface were collected from University farm (29°08′ 22.11″N; 75°42′ 16.92″E). Lesions were cut and sterilized with 0.1% HgCl2. The sterilant was removed through successive treatment with distilled water. Then, lesion spots were inoculated on potato dextrose agar (PDA) plates and incubated at 25 ± 2 °C in BOD incubator for 5–7 days. After sporulation, the morphological characteristics of spores were checked microscopically. The fungal cultures were stored at 4 °C in refrigerator for further use. 2.3. Screening of rhizobacterial isolates for antagonistic activity The antagonistic interaction of rhizobacterial isolates with Alternaria brassicae was studied by the spot test method (Sindhu et al., 1999) on modified LB medium plates. The fungus A. Brassicae was grown on PDA slants and spore suspension of the fungi was prepared in 3 ml sterilized water. Fungal spore suspension (200 µl) was spread over modified LB medium plates. Plates were incubated for 2 h in BOD incubator at 28 ± 2 °C. Bacterial growth suspension (2.0 μl) prepared from 48-h old growth of the rhizobacterial isolates was spotted on preseeded plates. Growth inhibition of fungi was recorded after 3 days of incubation at 28 + 2 °C. Antagonistic activity of the rhizobacterial
%Disease incidence(DI) =
2
No. of diseased plants × 100 Total no. of plants
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%Disease control No.of infected plants in control−No.of infected plants in treatment = No of infected plants in control
2.7. Statistical analysis Completely Randomized Design (CRD) was used for experimental data analysis. All determinations were carried out in triplicate and data represented are average values of three replications. Standard Error of Means ± (SEM) values were calculated to determine the significant differences between treatment means. The C.D. and C.V. values represent coefficient of deviation and coefficient of variation, respectively.
Fig. 1. Frequency of antagonistic isolates for fungal growth inhibition, Halo zone to growth ratio has been reported for 80 different rhizobacterial isolates.
(Table 2). More ALA production was observed in isolates HMR57 (19.95 µg ml−1) and HMR32 (16.33 µg ml−1). Out of 23 selected bacterial isolates tested, only six isolates HMR25, HMR29, HMR52, HMR60, HMR69 and HMR73 showed HCN production. Eight rhizobacterial isolates i.e. HMM49, HMM97, HMR25, HMR32, HMR52 HMR56, HMR69, and JMM16 showed siderophore production.
3. Results 3.1. Screening of bacterial cultures for growth inhibition of fungi All the 383 bacterial isolates were screened for their antagonistic interaction against fungi Alternaria brassicae on modified LB medium plates. The fungal growth inhibition zone varied with different bacterial isolates. Only, 80 isolates inhibited the growth of A. brassicae. Rhizobacterial isolates HMM51, HMR25, HMR33 and HMR70 showed fungal inhibition ratio of more than 4.0 (halozone to growth ratio) (Table 1, Figs. 1 and 2a, b). Thirty-one isolates showed > 3.0 halozone to growth ratio whereas thirteen isolates showed > 2.0 halozone to growth ratio of fungal inhibition. Thirty-two isolates comparatively showed less inhibition of the fungus and the ratio of halozone to growth varied between 1.0 and 2.0. Overall, only 20.88% isolates showed antagonistic activity against A. brassicae and rest did not inhibit the growth of fungi.
3.3. Coinoculation effect of Alternaria brassicae and rhizobacterial isolates for disease control and plant growth On the basis of variation in antagonistic activity and beneficial attributes, five rhizobacterial isolates i.e., HMR25, HMR48, HMR70, JMM16 and WHA64 were selected for inoculation effect on mustard plants seeds under pot house conditions. At 50 days of growth, coinoculation of fungi with rhizobacterial isolates JMM16 and HMR48 showed 140.0 and 96.9% increase in shoot dry weight as compared to uninoculated control, respectively (Table 3, Fig. 3). Inoculation with rhizobacterial isolates HMR70 and JMM16 both caused 107.1% increase in root dry weight as compared to uninoculated control. Disease control was 100% in HMR25 inoculated plants (Table 3). Rhizobacterial isolates HMR48 and WHA64 both showed 80.0% disease control in comparison to uninoculated control. At 75 days of growth, coinoculation of bacterial isolates HMR70, HMR48 and HMR25 with fungi showed 94.0, 77.5 and 95.5% increase in shoot dry weight as compared to control, respectively (Table 3, Fig. 3). Coinoculation of fungi and rhizobacterial isolates HMR70 and HMR48 showed only 14.8 and 17.5% increase in root dry weight in comparison to uninoculated control, respectively. Disease control was 100% in HMR25 inoculated plants (Table 3, Fig. 4) and inoculation of rhizobacterial isolates HMR70 and HMR48 both caused 83.33% disease control. It was observed that rhizobacterial isolates HMR70 showed significant increase in shoot dry weight at both the stages of plant growth (Fig. 5), and inoculation of isolates JMM16 and HMR48 caused significant gains in shoot dry weight. Coinoculation of all the rhizobacterial isolates with A. brassicae improved the shoot dry weight at 50 days of plant growth. However, only four rhizobacterial isolates i.e., HMR25, HMR48, HMR70 and JMM16 showed significant gains in plant dry weight at 75 days of plant growth. Inoculation with rhizobacterial isolate HMR25 caused 100% disease control at both the stages of plant growth, whereas inoculation with rhizobacterial isolates HMR48 andHMR70 showed 83.33% disease control at 75 days of growth as compared with respective control (Figs. 3 and 4). Thus, inoculation of rhizobacterial isolates HMR70 and JMM16 enhanced the plant dry weight of mustard as compared to uninoculated control plants at both 50 and 75 days of growth (Table 3, Figs. 3–5). In case of inoculation of fungi and bacteria, isolates HMR70, HMR48 and HMR25 showed significant increase in shoot dry weight in comparison to control. The inoculation of bacterial isolate HMR70 and fungi, increased shoot dry weight by 81.5 and 94.0% at 50 and 75 DAS, respectively. Whereas, inoculation of bacterial isolate HMR25 and fungi increased the shoot dry weight by 72.3 and 95.5% at 50 and 75 DAS, respectively. At 50 DAS, isolates HMR70, JMM16, HMR48, WHA64 and HMR25 showed 60.0, 40.0, 80.0, 80.0 and 100% disease control,
3.2. Growth promoting characteristics of rhizobacterial isolates Twenty-three selected antagonistic rhizobacterial isolates were analyzed for their plant-growth promoting activities like production of ALA, IAA, HCN and siderophore. Maximum IAA production (12.11 µg ml−1) was shown by isolate HMR48 followed by JMM16 (2.17 µg ml−1) and12 antagonistic isolates did not produce IAA Table 1 Halo zone to growth ratio of different rhizobacterial isolates. Rhizobacterial Isolates
Inhibition of A. brassicae (Halo zone to growth ratio)*
CP109 HMM21 HMM49 HMM97 HMR3 HMR25 HMR29 HMR32 HMR33 HMR39 HMR48 HMR52 HMR55 HMR56 HMR57 HMR59 HMR60 HMR69 HMR70 HMR73 JMM16 WHA64 WHA99
1.45 1.22 3.43 2.44 2.86 5.0 3.24 3.83 4.68 3.44 1.32 1.54 1.32 2.54 2.11 1.65 1.25 1.23 4.12 3.22 2.51 1.58 3.15
* Halo zone to growth ratio = Halo zone area (0.5 H2) Growth area (0.5 G2). 3
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Fig. 2. (a, b) Growth inhibition of A. brassicae by different rhizobacterial isolates. Table 2 Beneficial attributes of different rhizobacterial isolates. Rhizobacterial isolates
IAA (µg/ml)
ALA (µg/ml)
HCN productionδ
Siderophore productionα
CP109 HMM21 HMM49 HMM97 HMR3 HMR25 HMR29 HMR32 HMR33 HMR39 HMR48 HMR52 HMR55 HMR56 HMR57 HMR59 HMR60 HMR69 HMR70 HMR73 JMM16 WHA64 WHA99
0.99 0.24 1.65 0.96 0 0.36 1.77 0.09 0 0 12.11 2.68 1.80 0.33 0 1.02 0 0 1.47 1.02 2.17 1.20 1.17
8.12 14.74 14.45 13.53 9.66 8.74 14.66 16.33 10.29 17.99 10.53 10.37 10.12 11.58 19.95 12.03 0 10.37 8.66 10.74 12.87 10.91 11.58
− − − − − + 2+ − − − − 2+ − − − − + 2+ − 2+ − − −
− − + 2+ − 2+ − + − − − ± − 2+ − − − 2+ − − + − −
Table 3 Effect of single and mixed inoculation on mustard growth under pot-house conditions. Treatments
DAS
Root dry weight (mg/ plant)
Shoot dry weight (mg/ plant)
Disease incidence (%)
Disease control (%)
Soil
50 75 50 75 50 75 50 75 50 75
20 ± 1.00 42 ± 1.52 28 ± 2.51 68 ± 1.15 46 ± 1.52 74 ± 1.52 58 ± 1.52 108 ± 1.15 42 ± 1.15 85 ± 1.52
49 ± 1.52 78 ± 2.51 72 ± 1.52 178 ± 1.52 65 ± 0.57 134 ± 1.52 170 ± 1.15 382 ± 1.15 118 ± 1.00 260 ± 3.05
– – – – 71.42 85.71 – – 28.57 14.28
– – – – – – – – 60.0 83.33
50 75 50 75
58 88 45 72
188 276 156 235
± ± ± ±
1.52 1.52 1.52 0.57
– – 42.85 42.85
– – 40.0 50.0
50 75 50 75
49 ± 1.52 104 ± 1.52 41 ± 0.57 87 ± 1.00
152 316 128 258
± ± ± ±
1.15 2.64 2.51 1.52
– – 14.28 14.28
– – 80.0 80.0
50 75 50 75
47 70 32 58
± ± ± ±
1.00 1.15 1.52 1.15
132 252 119 194
± ± ± ±
1.15 0.57 1.15 1.15
– – 14.28 28.57
– – 80.0 66.66
50 75 50 75
40 82 34 75
± ± ± ±
1.52 1.00 1.00 0.57
126 271 112 262
± ± ± ±
1.00 1.00 1.00 1.15
– – 0 0
– – 100.0 100.0
50 75 50 75
4.080 3.68 5.821 2.80
– – – –
– – – –
Soil + RDF T2 + A. brassicae T2 + HMR70 T2 + A. brassicae + HMR70 T2 + JMM16 T2 + A. brassicae + JMM16 T2 + HMR48 T2 + A. brassicae + HMR48 T2 + WHA64
δ
On the basis of yellow orange halos after 2–5 days of incubation at 28 ± 2 °C, the production of siderophore by the rhizobacterial isolates was scored as: −: No production, +: Moderate production, 2+: high production. α The change in the color of the strips from yellow to orange red was observed for hydrogen cyanide production, by the method of Alstrom and Burns (1989). On the basis of change in the color of the strips from yellow to orange red after 2–5 days of incubation at 28 ± 2 °C, the production of HCN by rhizobacterial isolates was scored as: −: No production, +: Moderate production, ++: high HCN production.
T2 + A. brassicae + WHA64 T2 + HMR25 T2 + A. brassicae + HMR25 CD CV
respectively. At 75 DAS, rhizobacterial isolates HMR70, JMM16, HMR48, WHA64 and HMR25 showed 83.33, 50.0, 83.33, 66.66 and 100% disease control, respectively (Table 3).
± ± ± ±
0.57 1.15 1.52 1.52
3.91 5.04 1.90 1.25
Values given are average value of three plants. Disease incidence is the% of plants infected and disease control is the% reduction of diseased plants after inoculation with bacteria. The values of nodule fresh and shoot dry weight are calculated as per plant basis. C.D. and C.V. values represent coefficient of deviation and coefficient of variation, respectively. SEM (Standard Error of Means) values are represented as ( ± ).
4. Discussion Antagonistic microorganisms play a major role in microbial equilibrium and serve as powerful agents for biological disease control (Ana et al., 2009). The applications of biocontrol agents in the protection of some commercially important crops are increasingly capturing the imagination of many plant pathologists and microbiologists. In this study, 383 rhizobacterial isolates were studied for growth inhibition of A. brassicae on LB medium plates. It was found that only 20.88% isolates possess the ability to inhibit the pathogenic fungi A. brassicae in cultural conditions (Table 1, Figs. 1 and 2a, b).Foroutan et al. (2005)
obtained 132 isolates representing Pseudomonas and Bacillus species from the rhizosphere of wheat and thirty-two isolates showed antagonism against Fusarium graminearum. Bacillus megaterium isolated from tea rhizosphere produced siderophore and showed reduction in disease intensity (Chakraborty et al., 2010). To know the nature of antagonistic compounds produced by rhizobacterial isolates, HCN and siderophore production was examined in 4
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the selected rhizobacterial isolates. Isolate HMR25 showed siderophore production and also produced HCN (Table 2). Isolate JMM16 produced siderophore only. In similar studies, rhizosphere competent Mesorhizobium loti MP6 was found to produce hydrocyanic acid under normal growth conditions and enhanced the growth of Indian mustard (Brassica campestris) (Chandra et al., 2007). Jošić et al. (2015) reported that PGP isolates, Q16 and B25, showed the best antifungal activity against Trichoderma viride and good antifungal effect against Aspergillus fumigates and Aspergillus niger, while Penicillium verrucosum was the most resistant fungus. Three bacterial isolates HMR32, HMR39 and HMR57 showed more than 15 μg ml−1 ALA production (Table 2). Bacterial isolate HMR48 produced significant amount of IAA and ALA, and also inhibited the growth of A. brassicae. On the other hand, isolate HMR25 having highest antagonistic activity produced only ALA. Loper and Schroth (1986) observed a significant linear relationship between IAA accumulation of the rhizobacterial strains and decreased root elongation of sugar beet seedlings. Similar concentration dependent effects of IAA on stimulation or inhibition of root/shoot growth has been reported in earlier studies (Arshad and Frankenberger, 1991). Yaish et al. (2015) isolated bacterial strains that produced the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and the plant growth regulatory hormone indole-3-acetic acid. Some strains also chelated ferric iron (Fe3+), solubilized potassium, phosphorus and zinc, and also produced ammonia. Sarhan and Shehata (2014) tested four strains of plant growth promoting rhizobacteria (Bacillus subtilis, Paenibacillus polymyxa, Pseudomonas fluorescens and Pseudomonas putida) and Sinorhizobium meliloti for their antibiosis toward damping-off disease and yield of alfalfa crop. In vitro, the four PGPR strains produced hydrogen cyanide, indole-3-acetic acid, siderophore, solubilized insoluble phosphate and showed protease and β-1, 3-glucanaseactivities, whereas S. meliloti produced IAA and solubilized insoluble phosphate only. Such multi-trait rhizobacterial isolates having fungal growth inhibiting capability and plant growth promoting ability could have better potential for use as efficient biofertilizers and biopesticides under field conditions. In similar studies, various PGP bacteria were evaluated for plant
Shoot dry weight (mg/plant)
400 Control
350
HMR70
300
JMM16
250
HMR48
200
WHA64
150
HMR25
100 50 0 50
75 Days after sowing
Fig. 3. Coinoculation effect of rhizobacteria and fungi on shoot dry weight of mustard plants at 50 and 75 days of growth.
Fig. 4. Disease incidence (%) in case of different rhizobacterial isolates (DAS: Days after sowing).
Fig. 5. Growth of mustard plants inoculated with rhizobacterial isolates and/or fungi at 50 and 75 days. T2: Control + RDF; T3: T2 + A. brassicae. T4: T2 + Isolate HMR70; T5: T2 + HMR70 + A. brassicae. T6: T2 + Isolate JMM16; T7: T2 + JMM16 + A. brassicae. 5
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growth promoting (PGP) characteristics, biological control and rhizosphere competence (Bach et al., 2016). All bacteria showed competitive characteristics that could enhance rhizosphere competence either through hydrolytic enzyme production or through antagonistic activities. Both B. mycoides B38V and B. cepacia 89 showed remarkable antifungal activity in addition to other PGP characteristics, representing potential inoculants and biocontrol agents. Ayuke et al. (2017) studied the biological control of plant diseases through the addition of microbial biocontrol agents along with the earthworms as an environmentally friendly alternative to the chemical control of plant diseases. Beneficial bacterium Bacillus amyloliquefaciens and the earthworms Aporrectodea caliginosa or Aporrectodea longa were found to reduce the disease in oilseed rape (Brassica napus), when challenged with the pathogen Alternaria brassicae.
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5. Conclusion Eco-friendly agricultural system has emerged as an important thrust area globally for long-term soil environmental sustainability and to minimize the environmental pollution associated with indiscriminate use of chemical pesticides and fertilizers. Rhizosphere microorganisms are currently being explored for their possible use as biocontrol agents in the integrated pest management programmes as an environmentfriendly alternative intervention. Moreover, the capacity of living soil to sustain biological productivity using beneficial microorganisms has been improved recently by using various methodological developments in the last decades leading to promotion of plant health while maintaining environmental quality (Schloter et al., 2018). Keeping in view, the problem of environmental pollution and health hazards, microorganisms inhabiting the mustard rhizosphere have been identified in this study for the biological control of the Alternaria blight disease. Rhizobacterial isolates were found to inhibit the growth of Alternaria brassicae on LB medium plates. The inoculation of the selected rhizobacterial isolates i.e., HMR25 and HMR48 on mustard caused 55–114.6% increase in shoot dry weight and 80–100% disease control at 50 and 75 days of plant growth under pot house conditions. These results offer new opportunities to estimate the contribution of the rhizosphere bacteria under field conditions in the biological control of Alternaria blight along with promotion of mustard plant growth for sustainable agriculture. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsoil.2018.05.013. References Alstrom, S., Burns, R.G., 1989. Cyanide production by rhizobacteria as a possible mechanism of plant growth inhibition. Biol. Fertil. Soils 7, 232–238. Anal, E.P., Moreno, V.M., Cordo, C., 2009. Biological control of Septoria tritici blotch on wheat by Trichoderma sp. under field conditions in Argentina. Biocontrol 54, 113–122. Arshad, M., Frankenberger Jr, W.T., 1991. Microbial production of plant hormones. Plant Soil 133, 1–8. Ayuke, F.O., Lagerlöf, J., Jorge, G., Söderlund, S., Muturi, J.J., Sarosh, B.R., Meijer, J., 2017. Effects of biocontrol bacteria and earthworms on the severity of Alternaria brassicae disease and the growth of oilseed rape plants (Brassica napus). Appl. Soil Ecol. 117, 63–69. Bach, E., dos Santos Seger, G.D., de Carvalho Fernandes, G., 2016. Evaluation of
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