Evidence of inhibitory effect of Pseudomonas fluorescens CHA0 and aqueous extracts on tomato plants infected with Meloidogyne javanica (Tylenchida: Heteroderidae)

Effects of Pseudomonas fluorescens L. (jimsonweed) (Pf) isolate and the two plant extracts, Datura stramonium and Myrtus communis, were investigated on hatching and juvenile (J2s) mortality of Meloidogyne javanica(Tylenchida: Heteroderidae) under laboratory conditions. After determining the values of LC30, LC50, and LC70 of each extract, four leaf stage seedlings of tomato were treated by 20 ml of Pf suspension at a concentration of 108 CFU/ml, using a soil drenching method. After 1 week, the tested plants were inoculated by 4000 eggs and (J2s) of M. javanica and simultaneously were treated by 100 ml of the selected concentrations of D. stramonium (1.1, 1.4, and 1.8%) and M. communis (1.8, 3 and 5.2%), as soil drench. Results showed that a combination of Pf and the leaf extract, D. stramonium at the rate of 1.8% or M. communis at the rate of 5.2%, respectively, reduced the number of eggs per root system and the reproduction factor by 68 and 45%, the number of galls by 64 and 33%, and the number of egg masses by 65 and 43%, than the control. In conclusion, combination of Pf and D. stramonium at the rate of 1.8% or M. communis at the rate of 5.2% can significantly reduce the damage of M. javanica on tomato, under greenhouse conditions.


Background
Root-knot nematodes (Meloidogyne spp.) are among the most dangerous plant parasites (Jones et al. 2013) which cause a loss of 8.8 to 14.6% to agricultural products annually (Nicol et al. 2011). The nematodes have a short life cycle, a wide host range, and a high reproduction rate; therefore, their management is very difficult (Trudqill and Blok 2001). Besides, the application of chemical nematicides is harmful to the environment; their use also is not economically feasible. Therefore, due to their health consequences for human beings, application of chemical pesticides is limited and the researchers are searching for some safe and environmentally friendly methods which are based on economic and environmental issues. Some examples of such approaches are using plant extracts and herbal products like root exudates, herbal meal, and medicinal plant wastes (Marahatta et al. 2012). Biological control agents (Lamovsek et al. 2013;Silva et al. 2017) and their products (Radwan et al. 2012) are used in integrated pest management program.
Among the bio agents, plant growth-promoting rhizobacteria (PGPR)-such as Pseudomonas fluorescens CHA0-has a high efficiency in controlling plant pathogens like root-knot nematodes (Tavakol Norabadi et al. 2014). The outstanding feature of P. fluorescens is its high solubilization capacity of soil phosphorous (Galindo et al. 2018). Results showed that a mixture of P. fluorescens and Azospirillum brasilense had a positive influence on the yield of three potato varieties (Trdan et al. 2018).
In the last seven decades, plant extracts and other phytochemicals were surveyed for their effects on plantparasitic nematodes. Aqueous extracts of different parts of Neem, Chinaberry, and marigold were successfully used against the root-knot nematodes (Siddiqui and Shakeel 2007). Based on the chemical analysis of plant tissues from some plants, some chemical compounds have been detected. Marigold with alpha-terthienyl (Hethelyi et al. 1986) and Neem with azadirachtin, nimbin, salannin, nimbidin, and thionemone (Ferraz and de Freitas 2004) showed good effects on root-knot nematodes.
Considering the advantages of biological control, this study aimed to find out suitable aqueous extracts of some plants on juvenile mortality and hatching of M. javanica under laboratory conditions. The other target was to evaluate the efficiency of the combination of plant extracts and P. fluorescens CHA0 on reducing nematode damage in tomatoes greenhouse.

Materials and methods
Effects of different aqueous extract concentrations of Datura stramonium L. (jimsonweed), Myrtus communis L. (myrtle), Fumaria officinalis L., and Vitex agnuscastus L. (Chaste tree) on hatching and juvenile (J 2 s) mortality of M. javanica were analyzed under laboratory conditions. After identifying the suitable plant extracts, the necessary concentrations to cause 30, 50, and 70% J 2 s mortality were used in combination with an isolate of P. fluorescens CHA0 on tomato plants.

Preparation of root-knot nematodes culture
The roots of nematode-infected tomatoes were collected from greenhouses at Boyer-Ahmad County, Iran, and a single egg mass of the root-knot nematode, M. javanica, was cultured on tomato seedlings (cv. Early-Urbana) in the greenhouse. The root-knot nematodes species were identified based on the study of perennial pattern, as described by Taylor and Netscher (1974). In order to provide the suspension of nematode eggs, the method of Hussey and Barker (1973) was used. By storing the egg suspension in incubator adjusted to 27°C, J 2 s were hatched and were collected over a period of 4 days (Baghaee Ravari and Mahdikhani Moghaddam 2015).

Preparation of plant extracts
The aerial parts of the plants D. stramonium, M. communis, F. officinalis, and V. agnus-castus were collected from Boyer-Ahmad County, Iran, dried in shade and finely grinded using an electric grinder and a stock solution (10% w/v) was prepared (Ferris and Zheng 1999).

Preparation of bacterial isolate
The isolate of P. fluorescens CHA0 was obtained from the Department of Plant Protection, Faculty of Agriculture, Tehran University, Iran. To obtain a pure and fresh bacterial culture, bacterial suspension was grown on Nutrient Agar (NA) culture. The grown bacteria were harvested and mixed up with distilled water, and then the concentration was adjusted to 10 8 CFU/ml.

Laboratory assay
Two laboratory experiments were conducted to examine the inhibitory effect of plant extracts and bacterial suspension on J 2 s hatching and mortality in M. javanica. One milliliter of the egg suspension containing 100 ± 10 of nematode eggs was poured into the Petri dishes (with a diameter of 8 cm, then, 9 ml) of bacterial suspension with the concentration of 10 8 CFU/ml, or aqueous plant extracts at the rates of 0.5, 1, 2, 4, 6 and 8% (w/v) were added and then they were kept under controlled conditions at 27°C. After two periods of 72 and 120 h, the hatched juveniles were counted, using a stereo microscope (Gökhan and Sevilhan 2014). In another experiment, the J 2 s mortality was investigated. Nine milliliters of bacterial suspension with the concentration of 10 8 CFU/ml, or aqueous plant extracts at the rates of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, and 8% were added to 1 ml of nematode suspension containing 100 ± 10 J 2 s of M. javanica in Petri dishes and kept at 27°C, under controlled conditions. Dead J 2 s were counted with the help of a stereo microscope (Dourado et al. 2013). The experiments were carried out in completely randomized designs with four replications. The lethal mortality values (LC 30 , LC 50 , and LC 70 ) necessary to cause 30, 50, and 70% J 2 s mortality, were subjected to probit analysis in order to find the suitable concentrations.

Greenhouse experiment
Two greenhouse experiments were conducted at Boyer-Ahmad County, Iran, in 2017 and 2018. Different lethal concentrations (LC 30 , LC 50 , and LC 70 ) of D. stramonium and M. communis were chosen for greenhouse test. Seeds of susceptible tomato (cv. Early-Urbana) were sown in plastic pots with 1000 g of steam sterilized soil mixture of farm soil (sandy loam soil with electrical conductivity (EC) = 0.671 dS m −1 , pH = 7.45, contains 76% calcium carbonate, 52.9 mg/ kg phosphorus, 0.170 mg/kg of organic matter and 0.0987 mg/kg of organic carbon), cow manure and sand with a ratio of 1:1:2, respectively. The pots were kept under controlled conditions in the greenhouse with 16:8 h light to dark photoperiod and 27 ± 5°C. At four leaf stage, each one of the tomato seedlings was treated by 20 ml of a suspension of 10 8 CFU/ml of P. fluorescens CHA0 as soil drench. After 7 days, treated seedlings were inoculated simultaneously by 4000 eggs and J 2 s of M. javanica and soil-drenched with 100 ml/pot of selected concentrations of D. stramonium, viz. 1.1, 1.4, and 1.8% (w/v) and M. communis viz. 1.8, 3 and 5.2% (w/v). Sixty days after nematode inoculation, plants were harvested and plant shoot length as well as the fresh, dry weight of shoot, and fresh weight of root was recorded. Number of eggs, galls, and egg masses per root system and number of J 2 s per pot were counted, and the reproductive factor of the nematode was calculated as described by Sasser and Taylor (1978). Approximately 2 months after the first trial of the experiment, for the same procedure was done as the second trial of the experiment. The experiments were carried out in a completely randomized design test with five replications.

Statistical analysis
For the greenhouse experiment, data of plant growth parameters were subjected to a 4 × 2 × 2 (plant extracts × bacterial isolate × nematode) factorial analysis of variance (ANOVA) and the data of nematode population indices were subjected to a 4 × 2 (plant extracts × bacterial isolate) factorial analysis of variance (ANOVA) in a completely randomized design, using SAS statistical software (SAS Institute, Cary, NC). For assays of the inhibition of hatching and J 2 s mortality, data were subjected to oneway ANOVA. To normalize the data sets, prior to ANOVA analysis, expressed data as percentages were transformed to arcsin values (ArcSin√X) and only untransformed arithmetic means were presented. Where the F-test showed significance difference at p < 0.01, treatment means were compared using least significant differences (LSDs).

Results and discussions
Laboratory assay Application of P. fluorescens CHA0 significantly reduced the percentage of hatching of M. javanica and increased its J 2 s mortality than those treatments, which had no bacteria (Fig. 1). The results of D. stramonium and M. communis extracts on hatching (Fig. 2) and J 2s mortality ( Fig. 3) indicated that the increase in plant extract concentrations and numbers of hatched eggs decreased, while the numbers of dead J 2 s increased. The effects of V. agnuscastus and F. officinalis extracts were low; therefore, they were not chosen for greenhouse experiments. Toxicity lines were established for both of D. stramonium and M. communis, then LC 30 , LC 50 , LC 70 , and LC 90 were calculated (Table 1). The inhibitory effects of the plant extracts against the nematodes may be related to the presence of some metabolites in the plant. These chemicals can either affect the growth of J 2 s or kill them inside the egg or can dissolve the egg masses of root- knot nematodes (Adegbite and Adesiyan 2005). Some plant extracts may affect the behavior of nematodes, such as host finding capabilities and ultimately killing them. This type of response of juveniles and nematode eggs to plant extracts can be due to the differences in the type of plant metabolites (Zuckerman and Esnard 1994). Because of the variations which exist in antimicrobial compounds of plant extracts and essential oils, there are different mechanisms for their activities. Considering the combined activity, these compounds destroy the cell wall membrane and increase cellular permeability and ionic leakage. Following the breakdown of phospholipid molecules of cell wall, mitochondria, and membrane proteins, as well as decomposition of cytoplasm, cells are severely damaged and will die (Burt 2004).

Greenhouse experiment
Plants treated with the extract of M. communis at the rate of 5.2% (w/v) had the longest shoot and the highest shoot fresh weight. Non-significant differences were observed between these treatments and nematode noninoculated plants, which were treated by plant extract at the rate of 3% (w/v) in the case of shoot length. The highest shoot dry weight and root fresh weight were observed in nematode non-inoculated plants that treated with plant extract at the rate of 5.2% (w/v) along with bacterial incubation. The shoot dry weights had nonsignificant difference with the non-inoculated plants and with myrtle extract at the rate of 5.2% (w/v) ( Table 2). The number of eggs, galls, and egg masses per root system and the reproductive factor were significantly reduced in the bacterial-treated and non-treated tomatoes receiving 5.2% (w/v) myrtle extract. They had significant difference than other treatments, except bacterial-treated and non-treated plants containing 3% (w/v) myrtle extract. The lowest numbers of J 2 s were observed in the soil of bacterial-treated and non-treated tomatoes applied with 5.2% (w/v) myrtle extract. It had nonsignificant difference with bacterial-treated plants with 3% myrtle extract (Table 3).
The results of application of D. stramonium extract indicated that the longest shoots were observed in nematode non-inoculated plants, when treated with the plant extract at the rate of 1.8% (w/v) with and without P. fluorescens CHA0 incubation. They indicated non-significant difference with nematode inoculated and bacterial-treated plants with plant extract at the rate of 1.8% (w/v) and with nematode non-inoculated plants treated with plant extract at the rate of 1.4% (w/v) without P. fluorescens CHA0 incubation. The highest shoot fresh weight was observed in healthy plants that were treated with 1.8% (w/v) plant extract, with or without P. fluorescens CHA0 incubation. They indicated non-significant difference with bacterial-treated nematodeinfected plants along with plant extract at the rate of 1.8% (w/v) and also with treated healthy plants with plant extract at the rate of 1.4% (w/v) with or without bacterial incubation. The highest shoot dry weights were in healthy treated plants with 1.8% (w/v) concentration with and without bacterial incubation. They had non-significant difference with bacterial-treated and nematode-infected plants that treated  with plant extract at the rate of 1.8% (w/v). The highest root fresh weights were observed in healthy bacterialtreated plants with 1.8% (w/v) of plant extract (Table 4). Numbers of eggs per root system and the reproductive factor of nematode in bacterial-treated and non-treated tomato roots, receiving 1.8% (w/v) of D. stramonium extract, were significantly reduced than other treatments. The lowest numbers of J 2 s in soil and egg masses per root system were observed in the root of bacterial-treated and nontreated plants with 1.8% (w/v) plant extract. There were non-significant differences with bacterial-treated plants receiving 1.4% (w/v) plant extract. The lowest numbers of galls per root system were observed on the roots of bacterial-treated plants that were treated with 1.8% (w/v) plant extract. There were non-significant differences between those and bacterial non-treated plants with 1.8% (w/ v) plant extract ( Table 5). The positive effects of D. stramonium and M. communis extracts in reducing the damages caused by rootknot nematodes have been shown in several studies. The powder of D. stramonium at the rates of 75 g/kg of soil caused an improvement in okra growth indices and decreased the M. incognita infections (Hussain et al. 2011). It was indicated that D. stramonium extract contains large quantities of saponin and flavenoid and small quantities of tannin and alkaloid so it caused mortality  of J 2 s of M. javanica in laboratory conditions, and also, it caused a significant decrease in nematode indices and a significant growth improvement in infected melon (Umar and Ngwamdai 2015). Phytochemicals, such as saponins, tannins, flavonoids, alkaloids, phenols, and steroids, cause a significant decrease in reproductive factor as well as the numbers of galls and eggs of M. incognita, and they increase the amount of yield and plant growth indices. In a study carried out on ethanolic and aqueous extracts of D. stramonium, D. innoxia, and D. tatula, the ethanolic extracts had more effects on egg hatch inhibition and increase the J 2 s mortality in M. incognito.
Moreover, they reduced nematode infection but were uninfluential to plant growth indices (Babaali et al. 2017). It is also indicated that with an increase in the concentration of aqueous extracts of D. stramonium, the J 2 s mortality was increased (Sidhu et al. 2017), which is consistent with the results of the present study.
In previous studies, the antibacterial effects of myrtle extracts were analyzed (Mansouri et al. 2001;Amensour et al. 2010). The nematicidal properties of these antibacterial compounds have not been proven so far. In the study of Oka et al. (2012), the effects of aqueous extract and powder of M. communis on J 2 s mortality in M.  javanica in soil and the number of eggs per root system and gall index were investigated and the results proved the nematicidal activities of myrtle plant. Induced systemic resistance (ISR) in response to rhizospheric bacteria is one of the mechanisms of resistance against plant pathogenic nematodes, such as root-knot nematodes. Systematic resistance, caused by P. fluorescens CHA0, is attributed to the secondary metabolite, namely 2, 4-diacetylphloroglucinol (Siddiqui and Shaukat 2003). This kind of resistance which is caused by P. fluorescens CHA0 was reported in other studies as well. Rhizobacterial isolates have different mechanisms in inhibiting the life cycle of plant-parasitic nematodes (Siddiqui and Mahmood 1999). The production of hydrogen cyanide, ammonium, hydrogen sulfide, antibiotic, and volatile fatty acids are other inhibitory mechanisms of P. fluorescens CHA0. These toxic metabolites affected the reproductive rate of nematodes, therefore decreasing the number of nematodes (Siddiqui et al. 2006). Furthermore, the antagonistic fluorescent bacterial used against plant-parasitic nematodes, which are compatible with the rhizospheres, has low costs and has no adverse consequences for the environment.

Conclusion
The study indicated that aqueous extracts of D. stramonium and M. communis in combination with P. fluorescens CHA0 had a potential to decrease the reproduction of the root-knot nematode, M. javanica, and can improve the plant growth indices of infected tomatoes under greenhouse conditions.