Background

Root-knot nematodes, Meloidogyne spp., are one of the main pest groups causing serious crop losses in agricultural areas. Over 90 species of genus Meloidogyne were recorded (Jones et al., 2013), and the most common root knot nematode species are the following: Meloidogyne javanica, Meloidogyne incognita, Meloidogyne hapla, and Meloidogyne arenaria. The studies concluded that these nematodes can infect more than 3000 host plant species in agriculture (Jung and Wyss, 1999; Hussey and Janssen, 2002; Abad et al., 2003. Consequentially, among the many nematodes, the groups having some economic impact, Meloidogyne spp., are responsible for a large part of the annual 100 billion dollar losses attributed to nematode damage worldwide (Ralmi et al., 2016). The yield losses in vegetables such as tomato, melon, and eggplants exceed 30% (Sikora and Fernández, 2005).

The control strategies of plant parasitic nematodes in agriculture fields mostly include chemical, biological, physical, and cultural measures along with the use of resistant cultivars. The use of nematicides is not preferred because of environmental contamination and toxicity. Biological control of nematodes considered to be the best alternative to chemical control solely or in the Integrated Pest Management (IPM) concept. Biological control of plant parasitic nematodes involves mostly antagonistic fungi and bacteria. Most of endophytic bacteria can persist in the rhizosphere of many plant species, including vegetables and fruits without any adverse effect on overall plant health (Hallmann, 2001). Rhizobacteria could be used as biological control agents since they are colonizers of the root zone as soil microflora and sustain a positive effect on plant growth (Kloepper et al., 1992). Life cycle and development of most plant parasitic nematodes occur in the rhizosphere of host plants, where they closely interact with existing antagonists (Insunza et al., 2002). Rhizobacteria is categorized as plant health-promoting rhizobacteria (PHPR) (Sikora, 1988) or plant growth-promoting rhizobacteria (PGPR) (Kloepper et al., 1992). PGPR are free-living bacteria group that can colonize in the rhizosphere zone and stimulate root growth. The role of PGPR in the biological control concept was taken into consideration in some of recent studies. Albeit some Rhizobacteria species such as Bacillus sphaericus, Bacillus subtilis, and Pseudomonas fluorescens are reported to antagonize some of plant-parasitic nematodes (Tian et al., 2007), little information is available on the efficacy of PGPR bacteria on root-knot nematodes in vegetables.

This study was conducted to determine the effect of 15 PGPR strains against Meloidogyne incognita on tomato in the greenhouse.

Methods

Experimental design

The trials were conducted in a greenhouse located in Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey. The size of plastic pots used in the trials was about (2 L in capacity and 15 cm in diameter). The soil used consisted of 65% sand, 20% clay, 12% silt, and less than 3% organic matter. The trial was designed as a randomized complete block, consisting of 15 plant growth-promoting rhizobacteria (PGPR) strains (Table 1) and 2 controls: (−) without nematodes and bacteria together and (+) with nematodes but without bacteria. The experiment consisted of 10 replications with a total of 170 pots. Plant host used in the study was M. incognita-susceptible tomato, Solanum lycopersicum cv. 56-56 F1.

Table 1 The plant growth-promoting rhizobacteria (PGPR) strains used as treatments in the experiment
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Bacteria source and preparation

The bacterial strains were obtained from the bacterial collection in the Bacteriology Laboratory, Department of Plant Protection, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey. Vegetative cells of each bacterium were obtained by culturing them on nutrient agar medium (Merck, 1.05450) in disposable Petri dishes. They were kept at 25 ± 2 °C for 48 h; thereafter, each bacterial stain was suspended by saline buffer and arranged to 1 × 1010 cfu by spectrophotometer at 600-nm wavelength. Four-week-old tomato seedlings were treated by soaking in each of 15 bacterial cell suspensions individually for 10 min and transplanted promptly into the pots.

Nematode inoculum source

Meloidogyne incognita population used in this study was originally collected from field populations in Kahramanmaras. The nematode species was identified using perineal patterns and esterase phenotypes. A single egg mass was obtained from identified population reared on nematode-susceptible tomatoes in a greenhouse. In order to provide enough number of nematodes for the study, tomato plants were inoculated by the nematodes and reared in a greenhouse for 90 days. Then, the infected plants were cut at soil level, and the roots were washed under running tap water to remove all soil and debris. The needed number of nematodes was extracted from the harvested roots by Sodium Hypochlorite Extraction Method (Hussey and Barker, 1973). The obtained nematode suspension was sieved through double sieves by 75-μm mesh on the top and 25-μm mesh opening on the bottom. Extracted eggs and J2s were collected into a flask and kept in a refrigerator for 4 ± 1 days at 4 °C for further applications.

Nematode inoculation

Four days after transplantation into the pots, bacteria-treated four-week-old tomato seedlings were inoculated with 1000 eggs or J2/pot. Inoculation was preceded by forming four holes with 2–3 cm apart around the plant root. Each hole was covered by soil following the application of evenly distributed nematode inoculum. The experiment was completed in 90 ± 2 days. Over the course of the experimental period, all plants were irrigated regularly and fertilized with NPK (20-20-20) as needed.

Plant harvest

Ninety days after inoculation, the plants were cut off from crown part; the shoots were put in paper bags. Then, the plant roots were cleaned off from the soil by washing them gently by the tap water and keeping in the polyethylene bags in refrigerator for 4 ± 1 days at 4 °C for further processing.

Data collection

During plant growth, plants’ heights were measured and recorded biweekly. At harvest, root and shoot fresh and dry weight were recorded. At harvest, root, fresh, and dry weight, shoot fresh, and shoot dry weight were recorded. Root-galling indices were also calculated by using a 0–5 scale (Taylor and Sasser, 1978). The number of egg masses was recorded after the roots were stained by food coloring for 2 h (Thies et al., 2002), based on a 0–5 scale (Taylor and Sasser, 1978).

Data analysis

The data of plant growth parameters as well as a root-galling index and egg-mass indices were subjected to analysis of variance (ANOVA), and means were separated (P ≤ 0.05) by Duncan’s multiple-range test using SPSS, version 20.0.0. The root-galling indices and egg-mass indices were transformed to log(x + 1) before analysis.

Results and discussion

The all bacterial treatments influenced (P ≤ 0.05) all plant growth parameters as well as root-galling indices and egg-mass indices. There were significant differences of plant height among all treatments. The greatest plant height was recorded on the ZHA569-treated plants (59.03 ± 3.04 cm), while the lowest was in (+) control group (50.78 ± 3.70 cm). Also, the greatest root fresh weight was measured in ZHA90 (40.4 ± 9.49 g), and the lowest was in control (−) (21.1 ± 7.53 g). The greatest root dry weight was observed in ZHA90 (6.05 ± 2.35 g)-treated plants (Table 2).

Table 2 Comparison of means of plant height, root fresh weight, and root dry weight of Meloidogyne incognita-inoculated tomato plants, following bacterial treatments (mean ± SD)
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In general, the greatest and the lowest values for shoot fresh weights were recorded in the controls (−) (111.10 ± 25.37 g) and (+) (54.70 ± 10.78 g). While the highest shoot dry weight was observed in control (−) (18.29 ± 4.52 g), the lowest was in the strain ZHA569 (11.96 ± 3.74 g) (Table 3).

Table 3 Comparison of means of shoot fresh weight, shoot dry weight, root-galling indices and egg-mass indices of Meloidogyne incognita-inoculated tomato plants, following bacterial treatments (mean ± SD)
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Root-galling indices among treatments varied. However, the lowest root-galling indices were 0.678 ± 0.058 and 0.697 ± 0.042 for the strains ZHA296 and ZHA178, respectively. The lowest egg-mass indices were recorded at 0.727 ± 0.073 and 0.729 ± 0.059 for the strains ZHA296 and ZHA178, respectively (Table 3).

Different microorganisms such as endophytic bacteria and fungi could be utilized in biological control arena to protect plants against soil borne pathogens. The endophytic bacteria and fungi are able to colonize the rhizosphere zone and plant endorhiza and, consequently, can promote plant health against root-knot nematodes (Sikora et al., 2007). Plant growth-promoting bacteria could enhance plant growth and nutrition, therefore increasing plant resistance against pathogens (Compant et al, 2005 and Liu et al., 2012).

In the current study, Bacillus pumilus strain ZHA90 increased plant height, root fresh weight, root dry weight, shoot fresh weight, and shoot dry weight and reduced root-galling numbers. However, this strain did not affect egg-mass numbers. These results are aligned with those reported by Lee and Kim (2016) for M. arenaria on tomato. Another study by Mekete et al. (2009) revealed that B. pumilus reduced root-galling and egg-mass numbers of M. incognita on Ethiopian coffee. Bacillus megaterium reduced egg hatching and the number of second-stage juvenile (J2) of Meloidogyne incognita (Huang et al., 2010), which was likely resulted from nematotoxic compounds or extracellular hydrolytic enzymes of bacteria destroying nematode eggshell and juvenile cuticle.

In our study, ZHA296 (Paenibacillus castaneae) significantly affected egg-mass and root-galling indices and shoot dry weight. In another study, Paenibacillus spp. reduced the rate of egg hatching and J2 number of M. javanica, M. hapla, M. incognita, M. enterolobii, M. chitwoodi, and M. fallaxin vitro (Bakengesa, 2016). These results may be attributed to bacterial products such as antibiotics and secondary metabolites (Timmusk et al., 2005). Similarly, the study of Son et al. (2009) showed that among 40 strains of Paenibacillus spp. screened, P. polymyxa GBR-462 and GBR-508 and P. lentimorbus GBR-158 showed the strongest nematicidal activities and prevented M. incognita eggs from hatching. It can be elaborated that gelatinase and chitinase enzymes may contribute to the nematode inhibition (Jung et al., 2002).

Strain ZHA215 increased plant height, and strain ZHA178 reduced egg-mass and root-galling numbers significantly. Strain ZHA178 did not affect plant height, shoot dry weight, shoot fresh weight, root fresh weight, and root dry weight compared to control (+). It was reported that Paenibacillus spp. could cause phytotoxicity on plants depending on the concentration of bacteria and climatic conditions (Bakengesa, 2016).

Mycobacterium confluentis strain ZHA246 increased plant height significantly but did not affect other parameters. M. immunogenum strain ZHA17 increased plant height, shoot fresh weight, and shoot dry weight but did not affect root fresh weight, root dry weight, root-galling indices, and egg-mass indices. Tsukamurella paurometabola strain ZHA569 enhanced plant height but did not affect other parameters.

Conclusions

The soil contains plenty of beneficial bacteria, fungi, and other symbiotic organisms around plant roots. Bacteria can colonize in the rhizosphere zone or inner part of plant tissues and enhance plant growth resulting insignificant resistance to diseases by improving plant nutrition. In our study, ZHA90 (B. pumilus) increased plant growth and reduced root gall in tomato, and ZHA296 and ZHA178 (P. castaneae) decreased gall number and egg-mass numbers. On the other hand, ZHA17 (M. immunogenum) increased plant growth but not affected nematode-related parameters. The level of effectiveness of these strains on related parameters was significantly greater than other strains tested. These results suggest that Bacillus pumilus, Paenibacillus costume, and Mycobacterium immunogenum may be fairly good factors in suppressing the population density of M. Incognito tomato, despite of some discrepancy in the number of egg masses and the amount of root galling on the roots. These results also warrant additional long-term experiments with extended time to understand the dynamics of PGPR in field soil and to determine whether nematode population densities can be maintained at acceptable levels. Nevertheless, the results indicated that among 15 bacterial strains tested, ZHA296 and ZHA178 of P. castaneae and ZHA17 and ZHA57 of M. immunogenum could be used as promising biocontrol agents for the future nematode management strategies.