MOLECULAR CHARACTERIZATION AND ALLELOPATHIC MANAGEMENT OF Meloidogyne incognita (KOFOID AND WHITE) CHITWOOD IN TOMATO

Abstract

Root knot nematodes (Meloidogyne spp.) are important obligate parasites attacking many vegetables, fruits and ornamentals worldwide. Nematode populations from thirty commercial production fields of tomato of Malakand division in the Khyber Pakhtunkhwa province of Pakistan showed wide variations within and among species using the perineal pattern morphology and molecular tools. Three species viz., Meloidogyne incognita, Meloidogyne javanica and Meolidogyne areanaria were found either alone or co-infesting tomato roots (80.3%) and soil (87.3%). Disease was prevalent 100% with an average of 52.0% in the study area. More than one root knot nematode species were found together in the same plant roots; however, Meloidogyne javanica, occurred with the highest frequency (70.33%). A comprehensive molecular characterization of root knot nematode (RKN) populations belonging to ten localities of the Khyber Pakhtunkhwa province was carried out at the James Hutton Institute (JHI), Scotland, UK, employing the ribosomal DNA (rDNA) primers (D2A/D3B and 194/195) and species-specific SCAR primers i.e. Finc/Rinc (M. incognita), Fjav/Rjav (M. javanica) and Far/Rar (M. arenaria). Regardless of the species, the D2-D3 of 28S of rDNA gene and ITS2 region between 5S and 18S rDNA genes amplified the expected bands of approximately 750 bp and 720 bp, respectively common to all the populations tested. The SCAR primers generated species-specific bands of 1200, 670 and 420 bp in M. incognita, M. javanica and M. arenaria, respectively. Meloidogyne spp., were discriminated using mtDNA as an additional genetic marker. The C2F3/1108 primer pair amplified the COII/lrRNA region of mtDNA and produced a 1.7 Kb size band common to all the three species of RKNs except Meloidogyne chitwoodi (520 bp), Meloidogyne fallax (520 bp) and Meloidogyne enterolobii (750 bp), employed as negative control. Restriction digestion of the mtDNA-PCR product (1.7 Kb) with different 4-bp (Hinf 1, Taq 1, Mbo1, Alu 1) and 6-bp (Eco R1) restriction enzymes, amplified characteristic diagnostic patterns in each Meloidogyne spp., except the Taq1 enzyme which did not cleave the mtDNA-PCR product. The Hinf I generated three-banded diagnostic fragments (1700, 1300 and 400 bp) in M. incognita. The Mbo1 (viz., 1700, 1300, 1000, 720 and 520 bp) and Eco R1 (1700, 1200 and 520 bp) generated a five and three banded- pattern in all RKN populations respectively, whereas the Alu 1 enzyme produced frequent cuts in the mitochondrial genomes of all the three tested species. Genetic diversity among and within Meloidogyne species and populations were determined using the randomly amplified polymorphic (RAPD) DNA method. Three RAPD primers SC 10-30, OPG-13 and OPG-19 grouped the three mitotic species (Meloidogyne incognita, M. javanica, M. arenaria), in distinct separate cluster than the other species (M. chitwoodi, M. fallax, M. hapla and M. enterolobii) utilized as positive control. Meloidogyne javanica and M. arenaria were grouped more closely (50 %) than M. incognita (42.8 %). DNA sequencing ii of the 28S rDNA gene fragment of selected eight nematode genotypes (T1, W2, M3, J3, F2, J4, R2 and H1) belonging to three species (M. javanica, M. incognita, M. arenaria) representing the Khyber Pakhtunkhwa province were deposited to the Genbank with accession numbers (JQ317912-19). The intra-specific variability ranged from 3 nucleotides (0.4% differences) (between J3 and T1) to 27 nucleotides (4.2 % differences) (between W2, M3 and J3) for M. javanica (636 bp alignment). Sequence analysis of D2- D3 expansion segment of 28S rDNA did not discriminate the three closely related Meloidogyne spp. Juveniles and eggs of M. incognita were challenged in a series of in vitro experiments to plant extracts and pure compounds from a medicinal herb and annual weed, Fumaria parviflora Lam. The roots and stem crude extracts of the above plant showed the highest hatch inhibition (74.42 and 64.33%) and juvenile mortality (78.83 and 64.33%) against M. incognita at 12.5 mg mL-1. In in vitro experiments, the n-hexane extracts of the roots and stems showed the highest hatch inhibition (100%) and J2s mortality (100%). Hatch inhibition and J2s mortality were directly related to exposure time. The area under cumulative percentage hatch inhibition (AUCPHI) and mortality (AUCPM) were both augmented with increase in concentration. Silica gel column chromatography of the n- hexane and methanol fractions afforded eleven (F1 to F11) and seven (FM2.1 to FM2.7) sub-fractions, respectively. The F3 (98.77 %), F4 (90.25%) and FM2.1 (99.75%) exhibited the highest hatch inhibition at a concentration of 400 μg mL.-1 The J2s mortality for F3, F11, F4 and FM2.1 were 95.00, 88.25, 86.0 and 100%, respectively. The phytochemical screening of F. parviflora revealed the presence of seven classes of bioactive compounds (viz., alkaloids, flavonoids, glycosides, tannins, saponins, steroids and phenols). The quantitative determination of the plant extracts showed the highest percentage of alkaloids (0.9 ± 0.04) and saponins (1.3 ± 0.07) in the roots and total phenolic contents in the stem (16.75 ± 0.07 μg dry g-1). Three known nematicidal bioactive compounds viz., nonacosane-10-ol, 23a-homostigmast-5-en-3ß-ol from the roots n-hexane fraction and cis- and trans- prtopinium from the MeOH roots fractions of F. parviflora were isolated through activity-guided isolation. These compounds were identified through 1 H NMR and 13C-NMR, characterized and their physical properties determined. The 1H NMR and 13C NMR chemical shifts of cis-protopinium (minor) and trans-prtopinium (major) at 25 oC occurred in 2:1 and stability of trans-protopinium at 80 oC. Stem and root extracts of F. parviflora were evaluated for possible nematicidal activity against M. incognita in a screen-house trials. In pot trials with tomato, cv. Riogrande, F. parviflora roots and stem extracts, at concentrations of 1000, 2000 and 3000 ppm, applied as a soil drench, significantly reduced the root knot nematode number of galls, galling index, eggs masses, eggs and reproduction factor in comparison to the water control. Regardless of the concentrations, the application of all the extracts significantly increased the host plant parameters. The n-hexane extracts from the roots and stem were the most effective followed by methanol at all concentrations. In a second screen-house experiment, dried plant parts (roots, stem, foliage and the whole plant powder) of F. parviflora evaluated under varying application doses (0, 10, 20 and 30 g kg-1) significantly reduced the disease parameters. The number of galls and galling index, egg masses g-1 of roots, eggs per egg mass, adult root knot nematode females and J2 population were decreased substantially. The root powder dramatically reduced the galls (46.63 and 61.13), galling indices (2.33 and 2.96) and J2s populations (122.1, 250.7) in the spring and fall, 2010 in comparison to the control. The host plant growth parameters (shoot length, root length, fresh and dry shoot weight, number of branches plant-1 and number of flowers plant-1) increased significantly (P < 0.05). Field performance of amending soil with F. parviflora (roots, stems, foliage and whole plant) at different application doses (0, 10, 20 and 30 g dry powder plant-1) around the tomato rhizosphere showed positive plant responses in the spring and fall, 2010. Number of galls (31.00 and 39.25) and GI (1.25 and 1.87) were markedly reduced with the Fumaria roots powder. The fresh shoot weight (55.00 and 53.0 g), dry shoot weight (27.00 and 29.0 g), shoot length (48.0 and 55.0 cm), root length (21.50 and 26.75 cm), number of branches plant-1 (20.0 and 22.50 branches plant-1), number of flowers plant-1 (65.0 and 69.50) and number of fruits plant-1 (57.25 and 55.25) were significantly higher (P < 0.05) in the field treatments amended with the roots powder at the highest application dose. Conversely, the disease was severe in the untreated control plots which negatively affected the plant growth parameters. Results revealed that plant extracts, pure compounds and dry powder of F. parviflora can be used for management of root knot nematodes. Extracts and pure compounds of F.parviflora provide new insight for the development bio-commercial nematicides, in addition, F. parviflora shows great potential as a bionematicide because of the richness and diversity of compounds effective against Meloidogyne spp.