Algae material and preparation of ethanolic extracts
Samples of the red algae, Corallina mediterranea, C. officinalis and the green alga, Ulva fasciata Delile were provided by the National Institute of Oceanography and Fisheries (NIOF), Alexandria, Egypt.
The dried algae were powdered, using an electrical grinder, and 50 g of each algal powder were extracted in 500 ml of ethanol at 35 °C under regular stirring for 5 days. The extract was filtered through a Whatman No. 1 filter and evaporated to dryness in rotary vacuum evaporator (Unipan type 350 p, Poland) at 35 °C. The dried extracts were dissolved in ethanol to give 20 mg/ml stock solution, which was stored in airtight bottles at 4 °C and further diluted as per dose requirement.
Root-knot nematode culture and inoculum preparation
Root-knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood, inocula was prepared from a pure culture established from single egg mass and reared in eggplant (Solanum melongena L. cv. Black Beauty) which was grown in the greenhouse of the Faculty of Agriculture, Damanhur University, Egypt. Morphological characteristics of the female perineal patterns were used to identify M. incognita (Taylor and Sasser 1978). Eggs were extracted from the egg masses of the infected eggplant roots by sodium hypochlorite (NaOCl) solution (Hussey and Barker 1973), allowed to hatch in sterile distilled water at 25 ± 2 °C and newly hatched second-stage juveniles (J2) were collected for in vitro and pot experiments.
In vitro experiments
In vitro nematicidal activity of the three marine algal extracts, C. mediterranea, C. officinalis, and U. fasciata, at the concentrations of 0.125, 0.25, 0.5, and 1 mg/ml in a sterile distilled water were tested against M. incognita. For egg hatching test, 0.1 ml of water suspension containing 100 nematode eggs were transferred to glass vial containing 1 ml of double concentrations of tested extracts solutions and incubated at 25 ± 2 °C for 3, 5, and 7 days. After incubation, hatched juveniles were counted under light microscope (Olympus CX41RF, Olympus Optical Co., LTD) and egg hatching percentage was estimated.
The mortality of J2 was estimated by mixing 1 ml of water suspension containing 50 new-hatched J2 with 1 ml of double concentrations of tested extracts on glass vial and incubated at 25 ± 2 °C for 12, 24, and 48 h (Khan et al. 2016). After incubation, J2 were transferred in distilled water for 24 h, active and dead nematodes were counted by the microscope (Olympus CX41RF, Olympus Optical Co., LTD). The % mortality was calculated using Abbott’s Formula (Abbott, 1925) as follows:
$$ \mathrm{Juvenile}\ \mathrm{mortality}\ \left(\%\right)=\left(m-\mathrm{n}\right)/\left(100-n\right)\times 100 $$
Where m and n are percentages of dead juveniles in the treatment and control, respectively.
Glass vials containing 1 ml of a sterile distilled water contained either eggs or J2 suspension were served as control. The chemically synthesized nematicide, Oxamyl 24% SL (5 ml/l, DuPont Company, USA), was used for comparison. Five replicates with four vials per replicate were used for each concentration and the experiment was repeated three times.
Pot experiment
Surface-sterilized seeds of tomato (Lycopersicon esculentum Mill cv. Alisa) were germinated in seedling trays filled with a sterilized peat moss and allowed to grow up in the greenhouse for 4 weeks. Healthy and uniform 4-week-old seedlings were transplanted on plastic pots (one seedling per pot) of 20 cm diameter filled with 2.5 kg mixture of autoclaved sand and clay (3:1, v:v). Plants were kept on the greenhouse at 27–32 °C under natural daylight length conditions, watered once every 2 days, and fertilized once a week with commercial fertilizer with N (20%), P (20%), K (20%), and S (1.26%) (Vascon 20-20-20, Farmers for Agriculture Development, Egypt) at the rate of 2 g/l of water until the end of the experiment.
Ten days after transplanting, each pot was inoculated with 5000 eggs and freshly hatched J2. Ten milliliters of water suspension of eggs and J2 were pipetted into four holes of 5 cm depth around the plant roots zone and the holes were covered immediately with soil. Treatments included C. mediterranea, C. officinalis, and U. fasciata extracts at 1 mg/ml and Oxamyl 24% SL (5 ml/l) at recommended rate (5 ml/l) were applied as soil drench in 150 ml water per pot. All treatments were applied twice, after 2 days of transplanting and directly after inoculation. Untreated un-inoculated and untreated inoculated (nematode alone) pots served as controls. The experiment was arranged in a randomized block design with 20 replicates (pots) per treatment. Five replicates were used for nematode assay and the remaining replicates were used for enzymes and genes expression analysis. The experiment was repeated twice.
Plants were uprooted after 60 days of inoculation and gently washed with a running tap water, then shoot and root lengths (cm) and fresh weights (g) were recorded and the increase percentage over untreated inoculated control were estimated. Roots were stained in 0.015% Phloxine B solution for 15–20 min (Holbrook et al. 1983) and numbers of nematode galls, egg-masses, and eggs/plant root were counted and recorded (Taylor and Sasser 1978). Second stage juveniles (J2) were extracted and counted from 250 cm3 soil per pot using the bucket sieving technique (Cobb 1918). Reduction percentage in nematode parameters over untreated inoculated control was calculated as following: Reduction % = [(numbers in the control treatment − numbers in the treated plants) / number in control treatment] × 100. Reproduction factor (RF) was also calculated as following: (RF) = Pf/ Pi, where Pf = Final nematode population = number of eggs /plant + number of J2/pot at the harvest time and Pi = initial nematode population = 5000 eggs and J2.
Activity of the antioxidant enzymes
Peroxidase (POD) and polyphenol oxidase (PPO) were determined in tomato roots after 0, 2, 5, 10, and 18 days post inoculation (dpi). Three plants (replicates) per treatment were randomly collected at each specific sampling time, rinsed with demineralized water and stored at − 80 °C for further analysis. The same root samples were used for the quantification of gene expression at the same time points.
One gram of root was homogenized in 5 ml extraction buffer (50 mM phosphate buffer, pH 7.0, 1 mM EDTA, and 2% poly vinyl pyrrolidone, PVPP) in an ice-cold mortar. The homogenate was centrifuged at 12,000×g for 20 min at 4 °C (Universal 32R, Hettich Zentrifugen, Germany) and the supernatant was used for enzyme activity assays (Jakovljevicet et al. 2017).
POD and PPO activity assays were performed as described by Hussey et al. (1972). For POD activity assay, 0.1 ml of enzyme extract was incubated with 1.5 ml of 0.05 pyrogallol and 0.5 ml of 1% H2O2. The change in the absorbance was recorded at 20 s interval for 3 min at 420 nm (Jenway, Model 6305, Bibby Scientific Limited, UK). Enzyme activity was expressed as increase in the absorbance (Δ OD420) min−1 g−1 of fresh weight (FW) (Hammerschmidt and Kuae 1982). PPO activity was determined by adding 200 μl of enzyme extract to 1.5 ml of 0.1 M phosphate buffer (pH 6.5) and 200 μl of 10 mM catechol. The changes in absorbance were recorded for 1 min at 495 nm. Enzyme activity was presented as ΔOD495 min−1 g−1 FW (Mayer et al. 1965).
Expression analysis of the peroxidase and polyphenol oxidase and chitinase genes using real-time qPCR
Total RNA was isolated from root tissues sampled after 0, 2, 5, 10, and 18 dpi using GStract™ RNA isolation kit II (Maxim Biotech INC, USA) according to the manufacturer’s protocol. First-strand cDNA was synthesized from total RNA with oligo (dT) primer, dNTPS and M-MLV Reverse Transcriptase enzyme (Fermentas, USA) according to the standard protocol. QRT-PCR were performed for POD, PPO, and chitinase (Chit) genes. The tomato 18S rRNA gene was used as internal reference (housekeeping) gene (Nassar and Adss 2016; Jayanna and Umesha 2017).
The following primers were used in the RT-PCR reaction: POD (Jogaiah et al. 2013), F-5′-GCTTTGTCAGGGGTTGTGAT-3′, R-5′-TGCATCTCTAGCAACCAAC-3′, PPO (Goel et al. 2017), F-5′-CATGCTCTTGATGAGGCGTA-3′, R-5′-CCATCTATGGAACGGGAAGA-3′, Chit (Amaral et al. 2012), F-5′-AGTGCAGGAACATTCACTGGAGGA-3′, R-5′-ACACCAATACCTTGTCCAGCTCGT-3′, 18S rRNA (Jayanna and Umesha 2017), F-5′-GTGCATGGCCGTTCTTAGTTG-3′, R 5′-CAGGCTGAGGTCTCGTTCGT-3′. The RT-PCR Reaction (25 μl) contained 12.5 μl of 2× Quantitech SYBR® Green RT Mix (Fermentas, USA), 1 μl of 10 pmol/μl of each primer, 1 μl of template cDNA (50 ng), and 9.5 μl of RNase free water. The PCR reaction included an initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s, and a final extension of 72 °C for10 min (Chin et al. 2000). The reaction was performed using a Rotor-Gene 6000 (Qiagen, ABI System, USA). Relative quantification of gene expression was performed by (Δ C q = C q – reference gene, ΔΔ C q = C q – control, and ΔΔ C q expression = 2 (−ΔΔCq) (Livak and Schmittgen 2001). The expression levels of the target genes were normalized relative to 18S rRNA gene and relative expression of untreated control plants at each time were set as 1.
Statistical analysis
Data collected from in vitro and pot experiments were analyzed by two-way analysis of variance (treatments and times) using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). Treatment means were separated by Duncan’s multiple range test at 5% probability. Data of enzymes activity and gene expression were analyzed with GraphPad PRISM version 7 (GraphPad Software Inc., California, USA). Tukey’s HSD test was used to determine the significant differences between means at a probability level of ≤ 0.05.