Metabolic Bioproducts of Soil Streptomycetes and Their Effect on RNAi-Mediated Resistance of Tomato Plants Against Root-Knot Nematode Meloidogyne Incognita

Plant immunity and defense responses are putatively stimulated by the quality of light spectra. To determine whether aerial light quality can modify the resistance of tomato plants to root pests, in this study, tomato plants inoculated with Meloidogyne incognita were exposed to purple, blue, green, yellow and red light from light-emitting photodiodes at 20 µmol m−2 s−1 photosynthetic photon flux density at the level of the canopy during night for 4 weeks and the number of nematode galls (incidence), plant growth, and defense responses were investigated. Among the studied light treatments, only the nightly red (R) light treatment reduced the number of nematode galls in the roots; this was associated with a 7.9-fold increase in the expression of PROTEINASE INHIBITOR PROTEIN 1 gene, a 17.4-fold increase in the transcript of PATHOGENESIS-RELATED CLASS 1 (PR1) gene, and a 25.4 % increase in the salicylic acid (SA) content in the roots. Furthermore, nightly R light treatment significantly recovered nematode-induced decreases in the light saturated rate of CO2 assimilation and plant fresh weight; however, the other light treatments did not have similar effects. These results indicate that the exposure of the shoot to R light at night induces systemic resistance in tomato plants against root knot nematodes in the roots, which is partly dependent on the jasmonic acid and SA defence pathways. Our study also suggests that exposure to R light at night could be a potential approach to control the nematode infestation in tomato which is particularly feasible for greenhouse production.

The sustainable management of large amounts of fly ash (FA) is a concern for researchers, and we aim to determine the FA application in plant development and nematicidal activity in the current study. A pot study is therefore performed to assess the effects of adding different, FA-concentrations to soil (w/w) on the infection of chickpea plants with the root-knot nematode Meloidogyne incognita. Sequence characteristic amplified region (SCAR) and internal transcribed spacer (ITS) region-based-markers were used to molecularly confirm M. incognita. With better plant growth and chickpea yield performance, FA enhanced the nutritious components of the soil. When compared with untreated, uninoculated control (UUC) plants, the inoculation of M. incognita dramatically reduced chickpea plant growth, yield biomass, and metabolism. The findings showed that the potential of FA to lessen the root-knot nematode illness in respect of galls, egg-masses, and reproductive attributes may be used to explain the mitigating effect of FA. Fascinatingly, compared with the untreated, inoculated control (UIC) plants, the FA treatment, primarily at 20%, considerably (p ≤ 0.05) boosted plant growth, yield biomass, and pigment content. Additionally, when the amounts of FA rose, the activity of antioxidants like superoxide dismutase-SOD, catalase-CAT, and peroxidase-POX as well as osmo-protectants like proline gradually increased. Therefore, our findings imply that 20% FA can be successfully applied as a potential strategy to increase biomass yield and plant growth while simultaneously reducing M. incognita infection in chickpea plants.

Medicinal plant volatiles applied against the root-knot nematode Meloidogyne incognita

Genetic analysis of MIC-3 transgene with RKN resistance QTLs provides insight into the resistance regulatory mechanism and provides a framework for testing additional hypotheses. Resistance to root-knot nematode (RKN) (Meloidogyne incognita) in Upland cotton (Gossypium hirsutum) is mediated by two major quantitative trait loci (QTL) located on chromosomes 11 and 14. The MIC-3 (Meloidogyne Induced Cotton3) protein accumulates specifically within the immature galls of RKN-resistant plants that possess these QTLs. Recently, we showed that MIC-3 overexpression in an RKN-susceptible cotton genotype suppressed RKN egg production but not RKN-induced root galling. In this study, the MIC-3 overexpression construct T-DNA in the single-copy transgenic line '14-7-1' was converted into a codominant molecular marker that allowed the marker assisted selection of F2:3 cotton lines, derived from a cross between 14-7-1 and M-240 RNR, having all possible combinations of the chromosomes 11 and 14 QTLs with and without the MIC-3 overexpression construct. Root-knot nematode reproduction (eggs g(-1) root) and severity of RKN-induced root galling were assessed in these lines. We discovered that the addition of MIC-3 overexpression suppressed RKN reproduction in lines lacking both resistance QTLs and in lines having only the chromosome 14 QTL, suggesting an additive effect of the MIC-3 construct with this QTL. In contrast, MIC-3 overexpression did not improve resistance in lines having the single chromosome 11 QTL or in lines having both resistance QTLs, suggesting an epistatic interaction between the chromosome 11 QTL and the MIC-3 construct. Overexpression of MIC-3 did not affect the severity of RKN-induced root galling regardless of QTL genotype. These data provide new insights into the relative order of action of the chromosomes 11 and 14 QTLs and their potential roles in regulating MIC-3 expression as part of the RKN resistance response.

Root-knot and cyst nematodes are obligate plant parasites that induce complex biotrophic feeding structures in host roots. The mechanisms by which nematodes regulate host gene expression to produce feeding sites are unknown. The cauliflower mosaic virus (CaMV) 35S promoter has been reported to be repressed strongly in the feeding sites of both root-knot and cyst nematodes. In contrast, other work has indicated that this promoter is partially active in some feeding sites. Considering the importance of the 35S promoter in biotechnology, we have defined the nematoderesponsive nature of this promoter in more detail. Transgenic tobacco harboring various 35S-uidA constructs was assayed for β-glucuronidase (GUS) activity after infection by root-knot nematodes (Meloidogyne incognita) and cyst nematodes (Globodera tabacum subsp. tabacum). The entire 35S promoter (-343 to +8) was active in giant cells induced by M. incognita and, to a lesser extent, the syncytia of G. tabacum subsp. tabacum. In the latter case, activity decreased as the feeding sites matured. Subdomains of the 35S promoter were also active in feeding sites, particularly B4 and B5 in giant cells. However, subdomain B3 was strongly down-regulated in gall tissue and syncytia. In total, 14 constructs were studied and nematode-responsive expression was always stronger and more consistent with the root-knot nematode than the cyst nematode.

Root-knot nematode (Meloidogyne incognita) is one of the most destructive soil-borne pathogens that severely affect tomato (Solanum lycopersicum L.) production worldwide including Bangladesh, causing significant yield losses. The present study was conducted to evaluate the nematicidal potential of neem (Azadirachta indica A. Juss) based products for the management of M. incognita and their effects on tomato growth and yield under both pot culture and field conditions during 2017-18, 2018-19, 2019-20 and 2020-21 cropping seas. Different neem formulations, including neem seed extract, neem leaf extract, neem oil, and commercial neem cake, were applied artificial root knot nematode infested soil amendment. In pot house condition, all neem based product was effective against root knot nematode and increased plant growth parameters. In the field condition, results revealed that all neem products significantly reduced root-knot gall index, and increased plant growth parameters such as shoot and root growth as well as fruit number and yield of tomato compared to the untreated control. Among the tested treatments, neem oil cake and neem seed extract treatment exhibited the best nematicidal activity, with a reduction in gall index and remarkable improvement in plant height, shoot weight, root weight, fruit number, and total yield of tomato. Neem leaf extract, neem leaf powder and chemical nematicide Furadan 5G treatments also performed better in reduction of gall index, improving plant growth and yield of tomato compared to untreated control. These findings suggest that neem-based products, particularly neem oil cake and neem seed extract, are effective, eco-friendly alternatives to synthetic nematicides for sustainable root-knot nematode management and yield improvement in tomato production systems.

Poly[G] improved protein productivity of cell-free translation by inhibiting mRNase in wheat germ extract

We have demonstrated that mRNA, ribosome and resulting protein form complexes (ternary complexes) in wheat germ cell-free translation system and these complexes are stable for at least several hours. The protein folds into a proper conformation capable of specific binding with the inhibitor of its enzymatic activity. The removal of the stop codon from mRNA does not affect translation and mRNA-ribosome-protein complex stability. We have used these results to develop a method of isolation of mouse dihydrofolate reductase (mDHFR) encoding mRNA from native pool of mouse liver mRNA. The native pool of mouse liver mRNA was translated in vitro in a wheat germ cell-free translation system (WG-CFS), and enzyme-specific ternary complexes were affinity selected on a methotrexate-BSA coated 96-well microtiter plate (methotrexate, MTX, is an inhibitor of DHFR enzymatic activity). Bounded ternary complexes were eluted by MTX treatment. mRNA from eluates was amplified by template-switch RT-PCR and products of RT-PCR analyzed by gel electrophoresis. The cDNA was amplified by one-step reverse transcription-PCR and used for transcription, followed by translation and determination of the DHFR enzymatic activity in translation mixtures. This method is suitable for direct cDNA cloning from mRNA or cDNA libraries and for investigation of protein-protein interactions.

The content of nematicidal metabolites such as saponins, flavonoids and tannins in sulla (Hedysarum coronarium L.) suggests its potential nematicidal activity. In this study, the biocidal activity of 62.5–1000 μg mL−1 concentrations of flavonoid and tannin fractions from sulla was assessed in in vitro assays on the infective juveniles (J2) of the root-knot nematode (RKN) Meloidogyne incognita, while the suppressive effects of soil amendments with 10–40 g kg−1 soil rates of sulla biomass were investigated on potted tomato infested by M. incognita. The content of total nitrogen, carbon, flavonoids, tannins and saponins of sulla experimental material was also determined. After a 96-h exposure, more than 80% of the M. incognita J2 were killed even by a 125 µg mL−1 concentration of the flavonoid extract, while mortality peaked at 89% only at the 1000 µg mL−1 concentration of the tannin solution. Soil incorporation with sulla biomass significantly reduced the M. incognita densities both on tomato roots and in the soil, compared to either the non-treated control and chemical treatment with Fluopyram. The data confirmed the nematicidal potential of sulla, mainly due to its content of flavonoids and tannins, suggesting its suitability as green manure or a soil amendment for sustainable RKN management.

Two-way plant-mediated interactions between a plant parasitic nematode and a foliar herbivore arthropod

Upland cotton encounters biotic and abiotic stresses during the growing season, which significantly affects the genetic potential of stress tolerance and productivity. The root-knot nematode (RKN) (Meloidogyne incognita) is a soilborne roundworm affecting cotton production. The occurrence of abiotic stress (drought stress, DS) can alter the plant–disease (RKN) interactions by enhancing host plant sensitivity. Experiments were conducted for two years under greenhouse conditions to investigate the effect of RKN and DS and their combination using nematode-resistant (Rk-Rn-1) and nematode susceptible (M8) cotton genotypes. These genotypes were subjected to four treatments: control (100% irrigation with no nematodes), RKN (100% irrigation with nematodes), DS (50% irrigation with no nematodes), and DS + RKN (50% irrigation with nematodes). We measured treatments-induced changes in cotton (i) leaf reflectance between 350 and 2500 nm; and (ii) physiology and biomass-related traits for diagnosing plant health under combined biotic and abiotic stresses. We used a maximum likelihood classification model of hyperspectral data with different dimensionality reduction techniques to learn RKN and DS stressors on two cotton genotypes. The results indicate (i) the RKN stress can be detected at an early stage of 10 days after infestation; (ii) RKN, DS, and DS + RKN can be detected with an accuracy of over 98% using bands from 350–1000 nm and 350–2500 nm. The genotypes ‘Rk-Rn-1’and ‘M8’ showed differential responses to DS, RKN, and DS + RKN. With a few exceptions, all three stressors reduced the pigments, physiology, and biomass traits and the magnitude of reduction was higher in ‘M8’ than ‘Rk-Rn-1’. Observed impact of stressors on plant growth followed DS + RKN > DS > RKN. Similarly, leaf reflectance properties exhibited a significant difference between individual stress treatments indicating that the hyperspectral sensor data can be used to discriminate RKN-infected plants from drought-stressed plants. Thus, our study reveals that hyperspectral and physiological changes in response to RKN and DS could help diagnose plant health before visual symptoms.

Root-knot nematode disease caused by Meloidogyne incognita (Kofoid and White) Chit wood) is one of the major constraints for successful cultivation of tomato (Lycopersicon esculentum Mill.) in Ethiopia. Hence, the present study was conducted to evaluate the effect of leaf and seed extracts of four botanicals viz., Rape seed (Brassica napus L.), Lantana (Lantana camara L.), African marigold (Tagetes erecta L.) and Neem (Azadirachta indica L.) at 5% and 10% concentrations and T. harzianum at 5% plus control were tested on root-knot nematode under in vitro and also to evaluate their against root-knot nematode development and their role on plant growth parameters of tomato under in vivo condition. Plant extracts were more effective and significantly inhibited egg hatching and immobilizing the J2 larval mortality of M. incognita than T. harzianum. Aqueous extract of all the tested plants inhibited egg hatching of nematode and resulted 84.67-100% mortality of the J2 juveniles of M. incognita in vitro at the 10% concentration after 72 h of exposure time. There were no significant differences among the treatments of rape seed leaf (84.7%) at 10% concentration and Lantana camara (87%), African marigold (86.3%) and Neem leaf (85%) at 5% concentration after 72 h. Aqueous seed extracts of A. indica more significantly inhibited egg hatching and larva mortality of the J2 of M. incognita in vitro at the 10% concentration and immobilized by 89, 93 and 100% after 24, 48 and 72 h of exposures, respectively, while at similar concentration of T. erecta, B. napus and L. camara leaf extracts exhibited 92, 89 and 93.2% inhibition of egg hatching and 75, 62.1 and 73% larval mortality, respectively. The effect of different botanicals and T. harzianum singly and in combination were studied for the management of tomato root-knot nematode under greenhouse condition. There was a significant difference in the reduction of root-knot nematode incidence, root-knot nematode population, nematode reproduction rate (NRR), number of galls and egg masses per plant were recorded. In pot culture condition, the application of leaf extract of individual plant in the presence of the nematode significantly enhanced the growth of tomato seedlings in comparison to the control. A significant increase in plant height, shoot weight and root weight of the seedlings were observed at the 10% concentration of leaf extracts in comparison to control. There was a significant difference in the reduction of root-knot nematode population, nematode reduction rate, number of galls and egg masses per plant of L. camara combined with T. harzianum. The mean fruit weight and total yield were observed highest in the combination treatment of L. camara combined with T. harzianum. This study results revealed that the test plants are readily available to farmers at no cost and able to reduce nematode population below economic threshold.

The root-knot nematode (Meloidogyne incognita) is an important pathogen in crop cultivation, however, few methods are available to control this parasitic roundworm. In this study, the nematicidal effects of approximately 30 Streptomyces strains isolated from soil samples of Mt. Naejang (Korea) were tested against Meloidogyne incognita, and the culture broth of the strains KRA- 24 and KRA-28 exhibited approximately 75% and 85% insecticidal activity, respectively, in in vitro assays. In in vivo pot experiments, these strains reduced the number of nematodes in the soil and the number of egg masses in the roots of red peppers. The two strains also survived in the presence of insecticidal agents (0.1 to 3.0%) such as fosthiazate, ethoprophos and terbufos when they were used in parallel. The mixture of KRA-24 or KRA-28 culture broth and fosthiazate exhibited nematicidal effects that were similar to those observed when KRA-24 or KRA-28 were used alone. Our results clearly suggest that the Streptomyces strains KRA-24 and KRA-28 should be promoted as a biocontrol agent against Meloidogyne incognita.

Tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than L-tyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-L-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the L-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-L-tyrosine 10-fold more efficiently than L-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA(Tyr) was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-l-tyrosine. After the use of the 3-iodotyrosyl-tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of L-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-L-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

Fetal bovine pancreatic poly(A)-containing RNA directs the synthesis of an insulin immunoreactive polypeptide that is larger than proinsulin, preproinsulin, in the wheat germ cell-free translation system. We have characterized this peptide in detail both immunologically and chemically and have shown that it is 2500 daltons larger than bovine proinsulin (8700 daltons), possesses both insulin and bovine C-peptide-specific antigenic determinants, and contains all the tryptic peptides found in bovine proinsulin. Preproinsulin synthesized in the wheat germ cell-free system was precipitated with approximately 4-fold greater efficiency by bovine proinsulin antiserum than by insulin antiserum. Additional evidence was obtained which indicated that the preprotein folds and undergoes correct sulfhydryl oxidation less efficiently than proinsulin, perhaps due to the presence of the hydrophobic NH2-terminal extension. Automated sequential Edman degradation of bovine preproinsulin revealed the presence of an additional NH2-terminal sequence of 23 residues, preceding the B chain segment of proinsulin. The positions of 6 of the 7 leucine residues found in the bovine preproinsulin extension were identical to those reported previously for the rat preproinsulins. This close sequence similarity between the extensions of the bovine and rat preproinsulins supports the hypothesis that these molecules fulfill similar biosynthetic functions in vivo.