- Open Access
Evaluation of four rhizobacteria on tomato growth and suppression of root-knot nematode, Meloidogyne javanica under greenhouse conditions, a pilot study
© The Author(s) 2018
Received: 25 March 2018
Accepted: 27 June 2018
Published: 13 July 2018
Growth-promoting rhizobacteria are free-living bacteria that colonize the roots and stimulate the plant growth. Many of these bacteria secrete a range of extracellular metabolites that can be involved in the biological control of plant pathogens. In this study, the effect of four plant growth-promoting rhizobacteria (PGPR) was evaluated on tomato growth parameters as well as biological control of the root-knot nematode (RKN) (Meloidogyne javanica). Tomato seedlings were inoculated with four isolates of PGPR including Pseudomonas fluorescens, Poecilotheria striata, Bacillus subtilis, and Paenibacillus polymyxa and cultivated in the presence or absence of RKN. The results showed that the PGPR significantly increased the plant growth parameters. Bacterial strains also significantly affected the reproductive factor of Meloidogyne javanica significantly so that P. fluorescens and B. subtilis reduced the reproductive factor from 112.15 to 24.94 and 24.96, respectively. Based on these results, among applied rhizobacteria in this study, P. polymyxa can be regarded as the best candidate for promoting the growth and biological control of M. javanica in tomato crop under greenhouse conditions.
Tomato (Solanum lycopersicum L.) is an important crop in the world due to the diverse uses and high nutritional value of the fruit. In Iran, various species of root-knot nematode (RKN) have been reported from different crops including tomato, but the most prevalent one is Meloidogyne javanica (Damadzadeh 2007). Various microorganisms attack Meloidogyne spp. in soil and reduce their population of which the fungi, bacteria, and nematodes are the most important ones (Stirling 1991). A group of microorganisms that has been considered to improve plant growth and controlling RKNs are the plant growth-promoting rhizobacteria (PGPR) (Weller et al. 2002 and Lucy et al. 2004). Pseudomonas spp. are aerobic, Gram-negative bacteria, ubiquitous in agricultural soils, and are well adapted to grow in the rhizosphere (Weller 2007). Bacillus spp. have also an outstanding function in controlling plant pathogens and increasing plant growth parameters. Likewise other characteristics such as production of secondary metabolites, especially antibiotics, high tolerance to temperature alternations in the environment, rapid growth in vitro, and production of persistent endospores in the soil make Bacillus spp. as an appropriate option in controlling plant diseases (Backman et al. 1997). Bacillus thuringiensis decreased the populations of some important nematodes like Globodera pallida, M. javanica, and Meloidogyne incognita at remarkable levels (Racke and Sikora 1992 and Zukerman et al. 1993). Bacillus pumilus and Bacillus mycoides were the most effective bacteria in reducing the number of galls and egg masses of M. incognita by 33 and 39%; respectively (Mekete et al. 2009). PGPR exploit various mechanisms in suppression of RKNs. Pseudomonas aeruginosa could suppress the nematode activity by secretion of the enzymes proteinase and glycoproteinase (Ali et al. 2002). Bacillus cereus could decrease egg hatching of M. incognita up to 90% (Nagesh et al. 2005). Crude extracellular protein extract from culture supernatant of Brevibacillus laterosporus killed the nematode Panagrellus redivivus within 72 h (Huang et al. 2005). Culture filtrates of five isolates of Pseudomonas fluorescens and one isolate of each species of Pseudomonas putida, Bacillus subtilis, Brevibacillus brevis, and Serratia sp. caused 35–38% reduction in egg hatching and 32.2–48.8% increase in the mortality rate of juvenile two (J2) larvae of M. incognita (Behzadi Amin et al. 2014).
The objective of this research was to study the effect of four PGPR strains on plant growth parameters and on suppression of RKN (M. javanica) infecting tomato plants under greenhouse conditions.
Detection and preparation of nematode inoculum
In order to prepare the nematode isolate, a number of soil and root samples were collected from an infected tomato field in Kermanshah province, Iran. Single egg masses of root galls were picked up from well-washed infected tomato root and placed near 4-leaved tomato seedlings of the cultivar Early Urbana Y. These inoculated seedlings were maintained under favorable greenhouse conditions at 18–32 °C for 60 days to replicate nematodes. Identification of the pure population of Meloidogyne species was performed according to Taylor and Netscher (1974). Extraction of nematode from infected tissues for the experiments was performed according to Hussey and Janssen (2002) method.
Preparation of PGPR inoculum
Three strains of PGPR were received from Agrilife Biofertilizer Manufacturer, India, including Paenibacillus polymyxa (Prazmowski) Ash et al. (NCIM 2188), Bacillus subtilis (Ehrenberg) Cohn (MCC 0067), and Pseudomonas striata Chester (NCIM 2847). P. fluorescens (Flugge) Migula (333-S) strain was received from the Dep. of Plant Protection, College of Agriculture, University of Tehran, Karaj, Iran. Inocula of bacterial strains were prepared in Nutrient Broth (Quelab, Canada) on a shaker at 120 rpm for 48 h at 28–30 °C. Population density of the PGPR strains was measured with serial dilution method (Liddell and Parke 1989). Every PGPR strain had at least 108CFU/ml.
Effect of PGPR strains on plant growth parameters and on suppression of RKN
A factorial experiment was performed with 10 treatments and 4 replications in a completely randomized design. Bacterial factor had 5 levels (without bacteria, P. fluorescens, Paenibacillus polymyxa, P. striata, and B. subtilis), and RKN factor had 2 levels (zero population and 5000 eggs and larvae per kilogram of soil). Tomato seeds, cv. Falat, were sown in a pasteurized soil bed. Three weeks after cultivation, 4-leaved seedlings were transferred to the pots containing 1.5 kg 1:1 mixture of pasteurized soil and sand. Before planting, every seedling was inoculated by 1 ml of 108CFU/ml suspension of each bacterium. After transplanting, 5000 eggs and J2 larvae of M. javanica were added to every treatment (Hussey and Barker 1973). The pots were maintained at 18–32 °C and 16:8 day/night photoperiod for 60 days. To provide nutrients, the pots were irrigated with Hoagland solution every 2 weeks. At the end of the experiment, plant growth parameters including shoot height, root length, fresh and dried weight of shoot, and root and several indices related to nematode including mean number of galls and egg masses in the plant root, number of eggs in egg mass, and number of J2 larvae in soil were measured. The data were analyzed by SAS 9.4 software. Mean comparison of all factors were performed by least significant difference (LSD) at %1 probability level.
Results and discussion
Identification of the RKN
According to the cuticular perennial pattern of the female nematode body and morphological and morphometric detections of females and J2 larvae, the nematode species was confirmed as M. javanica (Hartman and Sasser 1985).
Impact of PGPR on plant growth parameters
The results of this research showed that applied PGPR not only promoted the plant growth parameters but also reduced the damage caused by M. javanica, which is in coordination with previous studies (Tian et al. 2007; Chauhan et al. 2015 and Amani Beni et al. 2016). However, there were insignificant differences among the bacterial treatments. The plants inoculated solely by M. javanica had the lowest shoot height. In the study performed by Moustaine et al. (2017), inoculation of several PGPR bacterial strains revealed the stimulatory effect of bacteria belonging to the genus Bacillus on the stem height and collar diameter of tomato plants. Root length data showed that PGPR improved root length either alone or in combination with nematode; however, there was insignificant difference among the four PGPR strains. Results of aerial parts fresh weight measurements indicated that P. fluorescens, P. polymyxa, and B. subtilis enhanced the shoot fresh weight in contrast to control significantly. The presence of PGPR in the treatments inoculated by both bacteria and nematode could minimize the reduction of the fresh weight of aerial parts, which in this sense, B. subtilis and P. polymyxa had the most effect in reduction of nematode damage and as a result increasing the fresh weight of aerial parts in these treatments. In the present study, P. striata increased the plant growth parameters and decreased the growth and development indices of the RKN, but compared to other treatments subjected to PGPR, the least effect was observed on plant growth and nematode damage reduction. In an experiment, seed inoculation of pearl millet [Pennisetum glaucum (L.) R. Br.] with P. striata improved the root and shoot biomasses and higher P uptake by straw and grain (Gaind 2013). Similarly combined application of Bradyrhizobium sp. with P. striata increased nodule occupancy in soybean resulting in more biological N2 fixation (Dubey 1996).
Comparison of the means of shoot dry weight revealed a significant difference between the healthy control, and the treatments inoculated by PGPR. B. subtilis and P. fluorescens had more effect to increase shoot dry weight.
Mean comparison of tomato growth parameters in treatments inoculated with plant growth parameters promoting rhizobacteria, P. fluorescens, P. polymyxa, P. striata, B. subtilis and root-knot nematode, and M. javanica under greenhouse conditions
Shoot length (cm)
Shoot fresh weight (g)
Shoot dry weight (g)
Root length (cm)
Root fresh weight (g)
Root dry weight (g)
P. fluorescens + M. javanica
P. striata + M. javanica
B. subtilis + M. javanica
P. polymyxa + M. javanica
Impact of PGPR to reduce the nematode contamination of tomato plants
Mean comparison of growth and developmental parameters of M. javanica on tomato plants inoculated and non-inoculated with plant growth promoting Rhizobacteria, P. fluorescens, P. polymyxa, P. striata, and B. subtilis under greenhouse conditions
No. galls/1 g root
No. egg masses/1 g root
No. eggs/egg mass
No. J2 /100 g soil
Reproductive factor (RF)
P. fluorescens + M. javanica
P. striata + M. javanica
B. subtilis + M. javanica
P. polymyxa + M. javanica
In this study, the mean number of J2 populations in the PGPR inoculated treatments was significantly lower than that of the infected control. Both of bacterial strains P. polymyxa and B. subtilis reduced J2 population to 129.5 comparing to 268 in infected control, which was equal to (51%) reduction of J2s in these two treatments (Table 2). PGPR exploit several mechanisms in suppression of plant pathogenic nematodes. Tian et al. (2007) showed that removal of alkaline protease BLG4 in Bacillus laterosporus resulted in destruction of 57% nematicidal activity of this bacterium. A neutral protease (npr) (designated Bae16) toxic to nematodes, which was purified from Bacillus nematocida could destroy the nematode cuticle and its hydrolytic substrates included gelatin and collagen (Niu et al. 2006).
Bacterial strains also significantly affected the reproductive factor of M. javanica, so that P. fluorescens and B. subtilis reduced the reproductive factor from 112.15 to 24.94 and 24.96, respectively (Table 2).
According to the results of this study, PGPR can be suitable candidates for use in biological control of M. javanica, which, in turns, will be an effective action to reduce the consumption of pesticides and helping to develop safer sustainable agriculture.
The authors are grateful to the Agricultural and Natural Resources Research Center of Kermanshah, Iran, for the facilities provided for conducting the study.
Availability of data and materials
The data and material are included in the dissertation of the first author but has not yet been published formally.
First and second authors, FS and MSH are responsible for conducting the experimental work. Second author MSH is responsible for designing and supervising the study, revising the paper scientifically, and checking analysis and interpretation of data. Third author, RH is responsible for supervising the study. Fourth and fifth authors, SR and RSH are responsible for the general cooperation and contribution to the study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Ali NI, Siddiqui IA, Shaukat SS, Zaki MJ (2002) Nematicidal activity of some strains of Pseudomonas spp. Soil Biol Biochem 34:1051–1058View ArticleGoogle Scholar
- Amani Beni F, Karegar A, Taghavi SM (2016) Effect of Pseudomonas fluorescens CHA0 and green manures of some inhibitory plants on activity of the root-knot nematode, Meloidogyne incognita and infected tomato growth parameters. Iran J Plant pathol 59(3):339–356Google Scholar
- Backman PA, Wilson M, Murphy JF (1997) Bacteria for biological control of plant diseases. In: Rechcigl NA, Rechcigl JE (eds) Environmentally safe approaches to crop disease control. CRC Lewis Publishers, Boca Raton, pp 95–109Google Scholar
- Behzadi Amin R, Kargar Bideh A, Taghavi SM (2014) Evaluation of rhizobacteria effects on the activity of root-knot nematode, Meloidogyne incognita under greenhouse and laboratory conditions. Iran J Plant Pathol 50(1):53–68Google Scholar
- Cetintas R, Kusek M, Ameen Fateh S (2018) Effect of some plant growth-promoting rhizobacteria strains on root-knot nematode, Meloidogyne incognita, on tomatoes. Egypt J Biol Pest Co 28:7View ArticleGoogle Scholar
- Chauhan H, Bagyaraja DJ, Selvakumarb G, Sundaram SP (2015) Novel plant growth promoting rhizobacteria, prospects and potential. Appl Soil Ecol 95:38–53View ArticleGoogle Scholar
- Damadzadeh M (2007) Agricultural nematology. Andisheh-gostar publisher, Isfahan, Iran, p 208 (in Persian)Google Scholar
- Dubey SK (1996) Combined effect of Bradyrhizobium japonicum and phosphate-solubilizing Pseudomonas striata on nodulation, yield attributes and yield of rain fed soybean (Glycine max) under different sources of phosphorus in Vertisols. Indian J Agric Sci 66(1):28–32Google Scholar
- Gaind S (2013) Pseudomonas striata for improving phosphorus availability in soil under pearl millet cultivation. J Crop Improv 27(3):255–271View ArticleGoogle Scholar
- Hartman KM, Sasser JN (1985). Identification of Meloidogyne species on the basis of differential host test and perineal pattern morphology. . In: advanced treatise on Meloidogyne Vol. 2. Raleigh NC. North Carolina State University pp 69–77Google Scholar
- Huang X, Tian B, Niu Q, Yang J, Zhang L, Zhang K (2005) An extracellular protease from Brevibacillus laterosporus G4 without parasporal crystal serve as pathogenic factor in the infection against nematodes. Res Microbiol 156:719–727View ArticlePubMedGoogle Scholar
- Hussey RS, Barker KR (1973) A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Dis Rep 57:1025–1028Google Scholar
- Hussey RS, Janssen GSW (2002) Root-knot nematodes: Meloidogyne species. In: Starr JL, Cook R, Brige J (eds) Plant Resistance to parasitic Nematodes. CABI, In, pp 69–77Google Scholar
- Khan Z, Kim SG, Jeon YH, Khan HU, Son SH, Kim YH (2008) A plant growth promoting rhizobacterium, Paenibacillus polymyxa strain GBR-1, suppresses root-knot nematode. Bioresour Technol 99:3016–3023View ArticlePubMedGoogle Scholar
- Liddell C, Parke J (1989) Enhanced colonization of pea taproots by a fluorescent pseudomonad biocontrol agent by water infiltration into soil. Phytopathology 79:1327–1332View ArticleGoogle Scholar
- Lucy M, Reed E, Glick BR (2004) Application of free living plant growth-promoting rhizobacteria. Antonie Leeuwenhoek 86:1–25View ArticlePubMedGoogle Scholar
- Mekete T, hallman J, Kiewnick S, Sikora R (2009) Endophytic bacteria from Ethiopian coffee plants and their potential to antagonize Meloidogyne incognita. Nematology 11(1):117–127View ArticleGoogle Scholar
- Moustaine M, Elkahkahi R, Benbouazza A, Benkirane R, Achbani EH (2017) Effect of plant growth promoting rhizobacterial (PGPR) inoculation on growth in tomato (Solanum lycopersicum L.) and characterization for direct PGP abilities in Morocco. Int J Environ Agric Biotechnol 2(2):590–596View ArticleGoogle Scholar
- Nagesh M, Asokan R, Mohan KS (2005) Partial characterization of novel nematicidal toxins from Bacillus cereus Frankland 1887 and their effect on root-knot nematode, Meloidogyne incognita ( Kofoid & White) Chitwood. J Biol Control 19(1):65–69Google Scholar
- Niu Q, Huang X, Zhang L, Li Y, Li J, Yang J, Zhang K (2006) A neutral protease from Bacillus nematocida, another potential virulence factor in the infection against nematodes. Arch Microbiol 185:439–448View ArticlePubMedGoogle Scholar
- Racke J, Sikora RA (1992) Isolation, formulation and antagonistic activity of rhizobacteria toward the potato cyst nematode Globodera pallida. Soil Biol Biochem 24:521–526View ArticleGoogle Scholar
- Seenivasan N, David PMM, Vivekanandan P, Samiyappan R (2012) Biological control of rice root-knot nematode, Meloidogyne graminicola through mixture of Pseudomonas fluorescens strains. Biocontrol Sci Tech 22(6):611–632View ArticleGoogle Scholar
- Stirling GR (1991) Biological control of plant parasitic nematode: progress, problems and prospects. CAB International, WallingfordGoogle Scholar
- Taylor DP, Netscher C (1974) An improved technique for preparing perineal patterns of Meloidogyne spp. Nematologica 20:268–269View ArticleGoogle Scholar
- Tian B, Yang J, Zhang KQ (2007) Bacteria used in the biological control of plant-parasitic nematodes: populations, mechanisms of action, and future prospects. FEMS Microbiol Ecol 61:197–213View ArticlePubMedGoogle Scholar
- Weller DM (2007) Pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years. Phtopathology 97(2):250–256View ArticleGoogle Scholar
- Weller DM, Raaijmakers JM, McSpadden Gardener BB, Thomashow LS (2002) Microbial populations responsible for specific suppression to plant pathogens. Annu Rev Phytopathol 40:309–348View ArticlePubMedGoogle Scholar
- Xie H, Pasternak JJ, Glick BR (1996) Isolation and characterization of mutants of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 that overproduce indole acetic acid. Curr Microbiol 32:67–71View ArticleGoogle Scholar
- Zukerman BM, Dicklow MB, Acosta N (1993) A strain of Bacillus thuringiensis for the control of plant-parasitic nematodes. Biocontrol Sci Tech 3(1):41–46View ArticleGoogle Scholar