In vitro Screening of Different Rhizobacterial Strains against Egg Hatching of Rice Root-Knot Nematode, Meloidogyne graminicola

Meloidogyne graminicola is an economic yield loss causing major pest of rice globally. For managing this pest, biological control practices act as an alternate strategy, as it is the most effective and economic. Plant growth promoting rhizospheric bacteria (PGPR) are most significant because they benefit plants both directly and indirectly by enhancing plant growth and provides long lasting antagonistic effects against plant parasitic nematodes. Considering this an in vitro efficacy research experiment of nematoxicity of native rhizobacterial strains was conducted, against egg hatching of M. graminicola at S/2 and S/4 concentration levels of intact bacterial culture and cell free culture filtrates (CFCs). The hatching behavior of rice root-knot nematode eggs was observed on alternate days for ten days which resulted in inhibition of egg hatching by all bacterial cultures at both concentration levels. When compared to the untreated control, Bacillus spp. showed the most egg inhibition in the S/2 concentration of both culture filtrates i.e,86.8% and 84.8%. Similarly, in the S/4 level of concentration, most egg hating such as 83.7% and 79.8% was recorded in Bacillus spp. culture filtrates. The rate of hatching was inversely proportional to the concentration of strains at exposure time, decreasing as the concentration increased. The results of the experiment revealed the potential of rhizospheric bacteria which makes it more feasible and environmentally safe approach for the management of Meloidogyne graminicola. &nbsp

Nematicidal Potential of Green Silver Nanoparticles Synthesized Using Aqueous Root Extract of Glycyrrhiza glabra

Meloidogyne incognita (root-knot nematode) is a devastating soil-borne pathogen which can infect almost all cultivated plants around the globe, expediting huge pecuniary losses. The purpose of current study was to use the aqueous root extract of Glycyrrhiza glabra for synthesizing silver nanoparticles (GRAgNPs) and assess their nematicidal potential against M. incognita by in vitro methods, including hatching inhibition and mortality assays. The active uptake of FITC labeled GRAgNPs by the nematode and their effect on the expression of selected genes involved in oxidative stress and DNA damage repair were also studied. An HRTEM micrograph confirmed their spherical morphology with sizes ranging from 9.61 nm to 34.735 nm. Complete inhibition of egg-hatching was observed after 48 h of treatment with as low as 10.0 ppm of GRAgNPs. In addition, 100% mortality was recorded at the lowest dose of 6.0 ppm, after 12 h of treatment. The LC-50 for GRAgNPs was found to be 0.805 ± 0.177 ppm at p < 0.0001, R2 = 0.9930, and α = 0.05. The expression of targeted genes (skn-1, mev-1, sod-3, dhs-23, cyp-450, xpa, cpr-1, gst-n, and ugt) was significantly enhanced (1.09–2.79 folds), at 1.0 ppm (α = 0.05, 95% CI) GRAgNPs treatment. In conclusion, GRAgNPs performed efficaciously and considerably in contrast to chemical nematicide and commercial silver nanoparticles (CAgNPs) and might be used as a promising alternative as relatively lower concentration and short exposure time were enough to cause higher mortality and nanotoxicity in nematodes

Utility of Host Delivered RNAi of Two FMRF Amide Like Peptides, <i>flp-14</i> and <i>flp-18</i>, for the Management of Root Knot Nematode, <i>Meloidogyne incognita</i>

<div><p>Root knot nematode, <i>Meloidogyne incognita</i>, is an obligate sedentary endoparasite that infects a large number of crop species and causes substantial yield losses. Non-chemical based control strategies for these nematodes are gaining importance. In the present study, we have demonstrated the significance of two FMRFamide like peptide genes (<i>flp-14</i> and <i>flp-18</i>) for infection and development of resistance to <i>M. incognita</i> through host-derived RNAi. The study demonstrated both <i>in vitro</i> and <i>in planta</i> validation of RNAi-induced silencing of the two genes cloned from J2 stage of <i>M. incognita</i>. <i>In vitro</i> silencing of both the genes interfered with nematode migration towards the host roots and subsequent invasion into the roots. Transgenic tobacco lines were developed with RNAi constructs of <i>flp-14</i> and <i>flp-18</i> and evaluated against <i>M. incognita</i>. The transformed plants did not show any visible phenotypic variations suggesting the absence of any off-target effects. Bioefficacy studies with deliberate challenging of <i>M. incognita</i> resulted in 50-80% reduction in infection and multiplication confirming the silencing effect. We have provided evidence for <i>in vitro</i> and <i>in planta</i> silencing of the genes by expression analysis using qRT-PCR. Thus the identified genes and the strategy can be used as a potential tool for the control of <i>M. incognita</i>. This is the first ever report that has revealed the utility of host delivered RNAi of <i>flps</i> to control <i>M. incognita</i>. The strategy can also be extended to other crops and nematodes.</p> </div

Bioefficacy analysis of the T₁ tobacco events against <i>Meloidogyne incognita</i>.

<p>Intensity of galling in the roots of T₁ tobacco plants expressing (A) <i>flp-14</i> (B) <i>flp-18</i> (C) Wild type.</p

Effect of host delivered RNAi of <i>flp-18</i> on infection and reproduction of <i>Meloidogyne incognita</i>.

<p>(A) Reduction in infection and reproduction of <i>Meloidogyne incognita</i> due to silencing of <i>flp-18</i> (B) Percentage reduction in infection and reproduction of <i>M. incognita</i> on transgenics compared to wild type plants. Error bars show mean +SD. Significant differences are marked with different alphabets, CRD test (_* P<0.05 and _** P<0.01).</p

Confirmation of dsRNA and siRNA of <i>flp-18</i> in T₁ plants by Northern analysis.

<p>(A) Northern blot for dsRNA; Lanes -NC: Negative control (Wild type tobacco plant), RNA samples from T₁ transgenic tobacco events 1- A.6, 2- A.30, 3- A.38, 4- A.39, 5- A.45, 6 - A.43, PC: Positive control (Gene specific PCR product). (B) Northern blot for siRNA, Lanes 1&2: <i>flp-18</i> T₁ tobacco events (A.45 & A.38), NC: Negative control (Wild type tobacco plant), PC: Positive control (Gene specific PCR product).</p

Percent reduction in the relative transcript levels due to target gene silencing in the females of <i>M</i>. <i>incogntia</i> feeding on the transgenic plants.

<p>(A) Decrease in mRNA abundance in <i>M. incogntia</i> females extracted from (A) <i>flp-14</i> (B) <i>flp-18</i> transgenic tobacco lines. Expression was quantified to demonstrate the target specific gene silencing in the nematodes through host delivered RNAi of <i>flp-14</i> and <i>flp-18</i>. <i>18S </i><i>rRNA</i> was used as a reference gene and fold change was calculated by using 2^-ΔΔCT method. Fold change values were transformed to personate values. Error bars show +SD among the biological replicates. ** P<0.01.</p

Effect of <i>in vitro</i> RNAi on transcript abundance of <i>flp-14</i> and <i>flp-18</i> in <i>Meloidogyne incognita</i> J2s.

<p>Expression was quantified to demonstrate target specific silencing in the nematodes. <i>18S </i><i>rRNA</i> was used as reference gene and fold change was calculated by using 2^-ΔΔCT method. The fold change values were transformed to personate values. Error bars show +SD among the biological replicates. **P<0.01.</p

Effect of <i>in vitro</i> RNAi on nematode attraction and penetration.

<p>(A) Nematode attraction to tomato roots at different time intervals (B) Penetration into tomato roots at 24 h (C) Percentage reduction in nematode penetration in tomato roots due to RNAi compared to control (D) Stained nematodes in the infected tomato roots (i) Roots inoculated with J2s soaked in water (control) (ii) Roots inoculated with J2s soaked in dsRNA of GFP (Unrelated control) (iii) Roots inoculated with J2s soaked in dsRNA of <i>flp-14</i> (iv) Roots inoculated with J2s soaked in dsRNA of <i>flp-18</i>. Error bars show mean +SD of the number of J2s that could be seen within 0.5 cm diameter around or inside the root. Significant differences are marked with different alphabets, CRD test (_* P<0.05 and _** P< 0.01).</p