Methods
Isolation and identification of nematode
Infected root samples were collected from a tomato greenhouse around the Urmia, West Azarbaijan province of Iran. The infected roots were isolated and propagated from a single egg mass of nematode on susceptible tomato cultivar, Super Strain B in greenhouse conditions with 27 ± 2 °C and 16:8 photoperiods.
Identification of nematode species was carried out using of perineal patterns of the females and subsequently confirmed with molecular identification using species-specific primers. The extraction of nematode genomic DNA was performed using modified method of Holterman et al. (2006). Polymerase chain reaction (PCR) was carried out in 10 μl solutions which consists of 4 μl of Ampliqon® 2X Master mix, 0.5 μM of each specific primers of M. incognita (Zijlstra et al. 2000), 100 ng of template DNA, and ddH2O. The reactions were carried out using Biorad® My Cycler TM as the following profile: one cycle of 4 min at 94 °C; 35 cycles including 94 °C, 30 s; 55 °C, 40 s; and 72 °C, 50 s; and a cycle of 7 min at 72 °C. The PCR products were loaded into agarose gel 1% that was stained by ethidium bromide. The results were observed in gel documentation (BioDocAnalyze, Biometra®, Germany) under long UV light.
Preparation, isolation, and identification of endophytic bacteria
In order to isolate of endophytic bacteria, samplings were performed from the multiple tomato farm soils. In laboratory, endophytic bacteria were isolated based on the Coombs and Franco (2003) method, using various parts of roots and shoots of the plants. Well sterilized and excised plant tissues were cultured in nutrient agar (NA) medium Liofilchem®, Italy, and incubated at 28 °C in incubator, Nabziran company, Iran. The resulted bacterial colonies were cultured and purified using single colonies. In this survey, 51 bacterial isolates were purified from different parts of plants of all samples. Twenty-two isolates were selected for biochemical identifications based on morphology of the colonies. In order to identify the bacterial isolates, some biochemical assays were performed (Hugh and Leifson 1953; Suslow et al., 1982 and Schaad et al. 2001). Gram staining, catalase, oxidase, lecithinase, and OF tests were among the conducted tests. The production of Levan, Indole, H2S, and the hydrolyses of casein, gelatin, starch, methyl red, ability of growth in 41 and 4 °C, tolerance in 5–7% NaCl, and HR on tobacco were the other tests for identification of the isolates.
Six selected bacterial isolates (1, 2, 5, 7, 10, and 11) were molecularly identified, using 16S rDNA sequencing. Extraction of bacterial genomic DNA was performed using the modified method of Omar et al. (2014). PCR tests were carried out in 50 μl solutions which consisted of 20 μl of Ampliqon® 2X Master mix, 2 μM of each primer of 16S rDNA region, 100 ng of template DNA, and ddH2O. The reactions were done using Biorad® My Cycler TM Thermal Cycler as the following profile: one cycle of 8 min at 94 °C; 40 cycles of 94 °C, 25 s; 55 °C, 45 s; and 72 °C, 30 s; and a cycle of 7 min (Baker et al. 2003 and Awad and Germoush 2017). The PCR products were loaded into agarose gel 1% that stained by ethidium bromide. The results were observed in gel documentation (BioDocAnalyze, Biometra®, Germany) under long UV light. The PCR products were sent for sequencing to Takapouzist Company, Tehran, Iran. The sequencings edited manually and were compared with relevant sequencings in NCBI data base using BLAST website. The sequence alignment was performed by ClustalW algorithm implemented in MEGA software v. 7.0.21. Evaluation of the evolutionary relationship of isolates and phylogenetic tree was generated by the neighbor-joining method.
Nematode mortality and egg hatching tests
The modified method of Siddiqui and Shaukat (2003) was performed for the tests. All of the bacterial isolates were cultured in tryptic soy broth (TSB), Liofilchem®, Italy. The cultures were incubated in incubator shaker (ISF1-X Climo-shaker, Kuhner shaker, Switzerland) in condition of 28 °C and 180 rpm. The bacterial media were centrifuged for precipitation and purified with distilled water. Water suspensions of 108 CFU/ml for all isolates of bacteria were prepared. Preparation of nematode egg suspension was done according to modified method of Hussey and Janssen (2002).
For evaluation of effects of bacterial isolates on hatching of nematode eggs, 1 ml of each suspension was added to wells of 24-well plates with four replicates. Four replicates of distilled water were considered as control. Hundred eggs of nematode were added in each well. The plates incubated at 26 ± 2 °C and the percentage of hatching were recorded on the 4th day after the experiment. For evaluation of impacts of bacterial isolates on mortality of second-stage juveniles (J2s), the suspension of juveniles was prepared. The eggs were isolated from the infected roots and transferred into Petri dishes containing sterile water and incubated in 27 ± 2 °C. Freshly hatched juveniles were collected for 3 days, and the resulted suspension was used for experiment. The isolates of bacteria and plates were prepared just alike the hatching experiment and finally 100 J2s added into the wells. The number of died juveniles was counted 1 to 4 days, and the percentage of mortality was recorded for each day. According to observed data, the 4th day data was selected for analyses. Both experiments were repeated twice, and the average of data was used for statistical analyses.
Greenhouse experiment
According to results of in vitro experiments, 6 primarily mentioned bacterial isolates (1, 2, 5, 7, 10, and 11) were selected for greenhouse experiments. Bacterial suspensions were prepared as described above. The suspension of eggs and juveniles of nematode was also prepared for inoculation of plants. The germinated seeds of tomato susceptible cultivar Super Strain B were cultured in trays containing autoclaved soil and perlite with 2:1 ratio. The trays were kept in a greenhouse with 27 ± 2 °C and 16:8 h photoperiod. Some of six-leaf stages of seedling were treated with bacterial suspensions and soaked in the prepared suspensions for 20 min and the others placed only in water. The seedlings were transplanted into the experimental pots containing 500 g of autoclaved soil and perlite with 2:1 ratio. Some of the treatments inoculated with 2000 eggs and J2s of nematode and the rest irrigated with water as control. The applied treatments on plants with selected and identified isolates were consisting of the following: A1 (B. wiedmannii), A2 (Pseudoxantomonas mexicana), A3 (P. thivervalensis), A4 (S. liquefaciens), A5 (P. chlororaphis), A6 (P. fluorescens), B1 (B. wiedmannii + nematode), B2 (P. thivervalensis + nematode), B3 (P. thivervalensis + nematode), B4 (S. liquefaciens + nematode), B5 (P. chlororaphis + nematode), B6 (P. fluorescens + nematode), C1 (nematode only), and C2 (without bacteria and nematode). The treatments were designed as complete randomized blocks with 4 replicates and kept in greenhouse with 27 ± 2 °C and 16:8 h photoperiod.
Data collection and analyses
Forty-five days after inoculation, some infection parameters of nematode on treated plants were recorded as number of galls and egg masses in root systems and per gram of roots and the number of eggs per egg mass. The gall and egg mass indices were assessed according to Taylor and Sasser (1978). The fresh and dry weights of roots and shoots and the height of shoots were also measured. Data analysis and drawing of graphs were performed using IBM® SPSS® statistics 20 and Microsoft® Excel 2013 software. The transformed data were exposed to analysis variance (ANOVA) and significant differences of the genotypes were realized using Duncan’s test.