Open Access

Stanzin Dorjey, Vishal Gupta*, V.K. Razdan and Richa Sharma

Division of Plant Pathology, Faculty of Agriculture, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, Chatha – 180 009, India.
J Pure Appl Microbiol. 2016;10(4):3079-3085
https://doi.org/10.22207/JPAM.10.4.81 | © The Author(s). 2016
Received: 17/08/2016 | Accepted: 03/10/2016 | Published: 31/12/2016
Abstract

Two major fungal pathogens, Fusarium oxysporum f. sp. lycopersici and Rhizoctonia solani were isolated from infected root, collar and stem regions of tomato plants.  A total of 25 rhizobacterial isolates showing Gram –ve reaction were isolated from the rhizosphere of tomato fields. Six selected isolates, based on their biocontrol activity against the test pathogens showed growth at 40C but there was no growth at 410C. The maximum growth of these isolates was at pH 7.0 and they isolates showed positive reactions for levan formation, phosphate solubilization, gelatin liquefaction, oxidase test and catalase test, that confirmed the identity of the isolates as Pseudomonas fluorescens. Under in vitro conditions, carbendazim showed 100 per cent inhibition of mycelial growth of F. oxysporum f. sp. lycopersici and R. solani at all the tested concentrations. Carbendazim also showed maximum plant vigour (1501.54 %), followed by isolate I-23 (1453.77 %).

Keywords

Soil borne disease, tomato, bio-control, management.

Introduction

Tomato (Solanum lycopersicum L.) is one of the most popular commercial vegetable crop grown throughout India and occupies an area of 8.65 lakh hactare, with annual production of 16.862 lakh MT1. There are several fungi, bacteria, viruses, nematodes and abiotic factors which adversely affect the productivity of tomato2. Out of different biotic stresses, Fusarium oxysporum f. sp. lycopersici and Rhizoctonia solani are the two most important soil borne pathogens, responsible for causing wilting or root rot in tomato crop. Management of soil-borne diseases has predominantly been based on chemical measures. However, overly dependence on chemical pesticides has not proved very effective, moreover, and their continuous use has proved hazardous by not only polluting soil and environment, but has also led to residual toxicity in soil and ground water. Fluorescent pseudomonads representing group of plant growth promoting rhizobacteria are known to promote plant growth and suppress plant pathogens by multiple mechanisms. Pseudomonas fluorescens is considered as putative biocontrol agent against various plant diseases including root diseases3 because of its production of secondary metabolites such as siderophores, antibiotics, volatile compounds, HCN, enzymes and phytohormones4. In order to develop an integrated disease management strategy based on eco-friendly measures, the present study was undertaken to explore the beneficial traits of P. fluorescens as biocontrol and plant growth promotion in tomato crop.

Materials and Methods

Isolation and identification of causal organisms associated with diseased plant samples
Isolation of fungal pathogens was done from infected plant parts such as root and/or stem/collor region of tomato plants5. Sterilized bits were asceptically transferred onto petriplates having pre-poured PDA + streptomycin sulphate and then incubated at 25+20C in BOD incubator. The petriplates were regularly observed for any fungal growth. The fungal growths obtained from diseased plant parts were purified using hyphal tip method6. Pure culture of each fungus thus obtained was used for further studies. Morphological examinations and cultural characteristics of the isolated fungi were recorded for their identification7,8. Pure cultures thus obtained were mass multiplied on Potato Dextrose (PD) broth for further studies.

Isolation and identification of rhizobacterial isolates
Rhizobacterial isolates were collected from the rhizosphere of tomato crop grown at  University Research Farm, by serial dilution agar plating method9. Aliquots from 10-7 dilution was placed in the centre of pre-poured petriplate containing King’s B medium. The inoculated plates were then incubated in inverted position for 24 hours in BOD incubator at 25+20C. Single colonies were purified by streak plate method10. Pure cultures were mass multiplied on nutrient broth medium and examined for their Gram reaction, colony morphology, fluorescence and cell shape11. Further, all the isolates were screened for bio-control potential against Fusarium oxysporum f.sp. lycopersici and Rhizoctonia solani by dual culture method12. Specific biochemical tests such as gelatin liquefaction, production of levan, catalase test, oxidase test, arginine hydrolysis, indole production, phosphorous solubilization and growth at 4 and 410C were conducted for identification of P. fluorescens13.

Effect of hydrogen ion concentration (pH)
To studying the effect of pH, the King’s B agar medium was amendment with varying hydrogen ion concentrations (pH) ranging from 3 to 14. The neutral pH of the King’s B agar medium was adjusted to acidic pH values by addition of 0.1N HCl and the alkaline pH by adding 0.1N NaOH before adding agar-agar and autoclaving. Sterilized petriplates were poured with 15-20 ml medium having different pH values and allowed to solidify. Then the plates were spotted with 10 ¼l of overnight cultures of the test organisms and incubated for 48 h at 28+20C. The observations on the ability of the isolates to grow at different hydrogen ion concentrations were recorded.

Evaluation of fungicides against the test pathogens
The efficacy of propiconazole (Tilt 25), tebuconazole (Folicur 25), hexaconazole (Malconda 5 EC) and difenoconazole (Score 25) at 10, 25 and 50 ppm concentrations, and carbendazim (Bavistin 50), mancozeb + carbendazim (SAAF), metalaxyl + mancozeb (Ridomil-MZ), triademifon (Bayleton) and carboxin + thiram (Vitavax Power) at 50, 100 and 250 ppm were evaluated in vitro, against the test pathogens i.e., Fusarium oxysporum f.sp. lycopersici and Rhizoctonia solani, using poisoned food technique14. Petriplates containing PDA amended with the desired concentrations of fungicides were inoculated with 5mm discs of individual pathogen and incubated at 25+20C. Petriplates without any fungicide served as check. The experiment was conducted under Completely Randomized Design with three replications. The radial growth of mycelium was recorded in each treatment and per cent inhibition over check was calculated15.

Evaluation of Pseudomonas fluorescens isolates for the management of soil borne disease of tomato
Potting soil (soil: FYM at 2:1) was autoclave-sterilized for 1 h on two consecutive days and was placed in pots. Seeds of tomato (Heem Sohna), treated individually with the selected isolates of Pseudomonas fluorescens (I-07, I-15, I-18, I-23, I-24 and I-25) were grown on nutrient agar medium for 48 hours and harvested with sterile distilled water (1×108 cfu/ml).  The bacteria coated seeds were spread on sterile filter paper, dried over night and sown in the pots. Thirty-day-old tomato seedlings pre treated with isolates of P. fluorescens as seed treatment were transplanted (4 seedlings pot”1) in earthen pots filled with sterilized potting soil. Ten days after transplanting, soil application with 10 ml of bacterial suspension (1×108 cfu ml”1) of the isolates (I-07, I-15, I-18, I-23, I-24 and I-25) was done and one day after soil application 50 ml of conidial suspension of Fusarium oxysporum f. sp. lycopersici (1000 microconidia ml”1) and  Rhizoctonia solani (8×105 cfu/g) was inoculated per pot. Further talc based bioformulation were also prepared by inoculation a loop full of P. fluorescens isolates into the nutrient broth16. Ten days after transplanting, soil application with talc based formulation (1g/pot) was done and one day after soil application, 50 ml of conidial suspension of F. oxysporum f. sp. lycopersici (1000 microconidia ml”1) and R. solani (8×105 cfu/g) was inoculated per pot. Carbendazim as seed treatment at 2g kg”1seed and after transplanting 2g pot”1as soil application was included as check, whereas, untreated seeds served as control. Observations regarding disease incidence and Vigour Index were recorded.

RESULTS AND DISCUSSION

Identification of plant pathogens
Two major fungal pathogens viz., Fusarium oxysporum f.sp. lycopersici and Rhizoctonia solani were found associated with the infected roots of tomato plants based on their cultural and morphological characteristic17, 18, 19 .

Identification and characterization of rhizboacterial isolates
Twenty five rhizobacterial isolates were obtained from the healthy tomato plants, (Table 1). Out of which, 14 were long rods and 11 were short rods. Majority of the isolates formed light green pigmented colonies (I-1, I-4, I-5, I-8, I-10, I-11, I-12, I-13, I-15, I-16, I-18, I-20, I-22, I-23, I-24 and I-25) whereas, the others formed green pigmented colonies (I-2, I-3, I-6, I-7, I-9, I-14, I-17, I-19 and I-21). Such a variation in the colony colour may be attributed to the production of different pigments/metabolites20. The isolates formed round to irregular shaped colonies, while the round shaped colonies were non-spreading, (I-1, I-4, I-5, I-8, I-10, I-11, I-12, I-13, I-15, I-16, I-18, I-20, I-22 and I-24) the irregular shaped colonies were of the spreading nature, (I-2, I-3, I-6, I-7, I-9, I-14, I-17, I-19, I-21, I-23 and I-25). All the 25 isolates exhibited fluorescence on King’s B agar under the UV light, however, there was variation among the isolates with respect to the intensity of fluorescence. The fluorescence under UV light is one of the key characters and the direct detection of fluorescence around the colonies is helpful for the identification of fluorescent pseudomonads21. Further, out of 25 isolates of fluorescent pseudomonads, I-07, I-15, I-18, I-23, I-24 and I-25 showed inhibition of pathogens viz., Fusarium oxysporum f.sp. lycopersici and Rhizoctonia solani (Table 2)

Table (1):
Cultural characteristics of rhizobacterial isolates

Isolate Colony characteristics Microscopic characteristics Fluorescens
Colour Shape Nature Cell shape Gram reaction
I-1 Light green Round Non-spreading Long rods Gram–ve ++
I-2 Green Irregular Spreading Long rods Gram–ve ++
I-3 Green Irregular Spreading Short rods Gram–ve +++
I-4 Light green Round Non-spreading Long rods Gram–ve +++
I-5 Light green Round Non-spreading Short rods Gram–ve ++
I-6 Green Irregular Spreading Short rods Gram–ve ++
I-7 Green Irregular Spreading Long rods Gram–ve ++
I-8 Light green Round Non-spreading Long rods Gram–ve ++
I-9 Green Irregular Spreading Short rods Gram–ve +++
I-10 Light green Round Non-spreading Long rods Gram–ve ++
I-11 Light green Round Non-spreading Short rods Gram–ve ++
I-12 Light green Round Non-spreading Short rods Gram–ve ++
I-13 Light green Round Non-spreading Long rods Gram–ve +++
I-14 Green Irregular Spreading Long rods Gram–ve +++
I-15 Light green Round Non-spreading Short rods Gram–ve ++
I-16 Light green Round Non-spreading Long rods Gram–ve ++
I-17 Green Irregular Spreading Long rods Gram–ve ++
I-18 Light green Round Non-spreading Long rods Gram–ve +++
I-19 Green Irregular Spreading Short rods Gram–ve +++
I-20 Light green Round Non-spreading Long rods Gram–ve ++
I-21 Green Irregular Spreading Long rods Gram–ve ++
I-22 Light green Round Non-spreading Short rods Gram–ve ++
I-23 Light green Irregular Spreading Short rods Gram–ve +++
I-24 Light green Round Non-spreading Long rods Gram–ve +++
I-25 Light green Irregular Spreading Short rods Gram–ve +++

++ =Good fluorescens; +++ =very good fluorescens

Table (2):
In vitro evaluation of rhizobacterial isolates against pathogens found associated with infected tomato plants

Isolate Fusarium oxysporum f. sp. lycopersi-ci Rhizoctonia solani
I-1
I-2
I-3
I-4
I-5
I-6
I-7 + + + +
I-8
I-9
I-10
I-11
I-12
I-13
I-14
I-15 + + + +
I-16
I-17
I-18 + + + +
I-19
I-20
I-21
I-22
I-23 + + + +
I-24 + + + +
I-25 + + + +

+ Inhibition; – Non-inhibition

Identification of Pseudomonas fluorescens
The six putative rhizobacterial isolates having bio-control potentials were identified based on biochemical test viz., levan formation, arginine hydroysis, indole production, phosphorous solubilization, gelatin liquefaction, oxidase test and catalase test (Table 3) which confirmed the identification of the selected isolates (I-7, I-15, I-18, I-23, I-24 and I-25) to be P. fluorescens 22. All the isolates showed positive growth at 40C, whereas, there was no growth at 410C. Earlier workers have also reported the similar results23. For physiological characterization, the effect of pH on growth was also studied and all the selected isolates showed maximum growth at pH 7.0, whereas, no isolate could grow at extreme acidic pH of 3.0 and 4.0 or alkaline pH of 13.0 and 14.0 (Table 4). All the isolates were able to grow in the pH range of 5.0 to 12.0.  The selected isolates showed very good to good growth at pH 6.0 to 9.0.24 isolated and characterized 216 fluorescent pseudomonads with high phosphate solubilizing ability from alkaline and Ca-rich soils with low phosphate availability. These reports indicate the ability of the fluorescent pseudomonads to sustain higher pH and NaCl concentration in the soil.

Table (3):
Biochemical characteristic of Pseudomonas fluorescens isolates

Biochemical tests Pseudomonas fluorescens isolates
I-7 I-15 I-18 I-23 I-24 I-25
Growth at 40C + + + + + +
Growth at 410C
Levan formation + + + + + +
Arginine hydrolysis + + + + + +
Indole production + + + + + +
Phosphorous solubilization + + + + + +
Gelatin Liquefaction + + + + + +
Oxidase test + + + + + +
Catalase test + + + + + +

Table (4):
Effect of hydrogen ion concentrations (pH) on growth of Pseudomonas fluorescens isolates

Isolate Growth at different pH levels
3 4 5 6 7 8 9 10 11 12 13 14
I-7 + ++ +++ +++ ++ + + +
I-15 ++ ++ +++ +++ ++ + + +
I-18 + ++ +++ +++ ++ ++ + +
I-23 ++ +++ +++ +++ +++ ++ ++ +
I-24 + ++ +++ ++ ++ + + +
I-25 ++ ++ +++ +++ ++ + + +

– Absent; + Fair; ++ Good; +++ Very good

Table (5):
In vitro evaluation of fungicides against the test pathogens

Fungicide Conc. (ppm) Radial growth (mm) Per cent inhibition over control
Fol Rs Fol Rs
Propiconazole (Tilt) 10 25.36 19.50 71.82 78.33
25 20.53 15.60 77.18 83.33
50 14.10 11.63 84.33 87.07
Tebuconazole (Folicur) 10 18.40 8.50 79.56 87.07
25 11.70 0.00 87.00 100.00
50 0.00 0.00 100.00 100.00
Hexaconazole (Malconda) 10 26.73 23.43 70.30 73.96
25 23.73 22.80 73.63 74.67
50 14.20 16.10 84.22 82.11
Difenoconazole (Score) 10 48.50 63.43 46.11 29.52
25 36.93 57.10 58.97 36.56
50 30.83 39.80 65.74 55.78
Carbendazim (Bavistin) 50 0.00 0.00 100.00 100.00
100 0.00 0.00 100.00 100.00
250 0.00 0.00 100.00 100.00
Triademifon (Bayleton) 50 59.63 68.06 33.74 24.38
100 47.96 57.86 46.71 35.71
250 36.13 42.70 59.86 52.56
Mancozeb + Carbendazim (SAAF) 50 19.16 13.76 78.71 84.71
100 14.13 23.60 84.30 73.78
250 8.87 0.00 90.14 100.00
Mancozeb + Metalaxyl (Ridomil-MZ) 50 22.03 25.93 75.52 71.18
100 17.90 21.10 80.11 76.56
250 12.13 11.60 86.52 87.11
Carboxin+thiram (Vitavax power) 50 61.80 22.80 31.33 74.67
100 52.03 14.00 42.18 84.44
250 41.83 9.36 53.52 89.60
Control 90.00 90.00 0.00 0.00
CD (P=0.05) 2.542 3.394
S.Em (+) 0.894 1.194

Fol = Fusarium oxysporum f.sp. lycopersici; Rs = Rhizoctonia solani

Evaluation of fungicides against the test pathogens
While evaluating the fungicides at different concentrations against F. oxysporum f. sp. lycopersici and R. solani, it was observed that all the treatments significantly reduced the mycelial growth of the test pathogens (Table 6). However, carbendazim, at all the tested concentrations and tebuconazole at 50 ppm, were responsible for complete inhibition of F. oxysporum f. sp. lycopersici, whereas, carbendazim (50, 100 and 250 ppm), tebuconazole (25 and 50 ppm) and mancozeb + metalaxyl (250 ppm) resulted in complete inhibition of R. solani. Our results are in accordance with25 who tested Captan, Emison-6, Foltaf, JKstein, Kavach, Shield-75 and Vitavax against R. solani, the causal agent of damping off in eggplant under in vitro conditions and concluded that JKstein and Vitavax were the most effective fungicides, whereas, Sheld-75 was least effective in checking the linear growth of the fungus. Carbendazim and benomyl were the most effective in vitro fungitoxicants against Fusarium spp. compared to captafol, thiram, thiophenate methyl and captan26. The efficacy of carbendazim and carboxin against R. solani under in vitro conditions has also been advocated by other researchers27, 28.

Table (6):
Effect of seed treatment and talc based formulation of Pseudomonas fluorescens isolates for the management of soil borne disease of tomato

Isolate Vigour index (%) Disease incidence %
Fusarium oxysporum f.sp. lycopersici Rhizoctonia solani
I-7 1212.62 12.50 29.16
I-15 1120.54 16.67 27.08
I-18 1253.82 27.08 10.41
I-23 1453.77 14.58 16.67
I-24 1299.70 25.00 18.75
I-25 1317.27 20.83 27.08
I-7+ Talc 1193.06 22.91 29.16
I-15+ Talc 1265.95 18.75 33.33
I-18+ Talc 1220.90 22.91 25.00
I-23 + Talc 1386.93 18.75 18.75
I-24 + Talc 1157.31 25.00 25.00
I-25 + Talc 1291.43 29.17 29.17
Talc alone 458.20 64.58 64.58
Carbendazim 1501.54 8.33 14.58
Control 453.70 72.91 62.50
S.Em(+) 41.97 2.21 2.90
CD(P=0.05) 121.81 6.41 8.43

Evaluation of Pseudomonas fluorescens isolates for the management of soil borne disease of tomato
The selected P. fluorescens isolates (I-7, I-15, I-18, I-23, I-24 and I-25) and carbendazim were tested in pot culture against F. oxysporum f. sp. lycopersici and R. solani. The fungicide carbendazim showed maximum plant vigour (1501.54%) followed by isolate I-23 with 1453.77 per cent vigour index (Table 7). Similarly29 have also reported that P. fluorescens increased plant vigour and consistently reduced the disease incidence under green house conditions and the disease protection was comparable with fungicide carbendazim.

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