Effects of different aqueous extract concentrations of Datura stramonium L. (jimsonweed), Myrtus communis L. (myrtle), Fumaria officinalis L., and Vitex agnus-castus L. (Chaste tree) on hatching and juvenile (J2s) mortality of M. javanica were analyzed under laboratory conditions. After identifying the suitable plant extracts, the necessary concentrations to cause 30, 50, and 70% J2s 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, J2s 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 108 CFU/ml.
Laboratory assay
Two laboratory experiments were conducted to examine the inhibitory effect of plant extracts and bacterial suspension on J2s 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 108 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 J2s mortality was investigated. Nine milliliters of bacterial suspension with the concentration of 108 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 J2s of M. javanica in Petri dishes and kept at 27 °C, under controlled conditions. Dead J2s 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 (LC30, LC50, and LC70) necessary to cause 30, 50, and 70% J2s 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 (LC30, LC50, and LC70) 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 108 CFU/ml of P. fluorescens CHA0 as soil drench. After 7 days, treated seedlings were inoculated simultaneously by 4000 eggs and J2s 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 J2s 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 J2s mortality, data were subjected to one-way 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).