Reprodução de Meloidogyne incognita raça 1 e Meloidogyne javanica em linhagens avançadas de tomateiro industrial.

2009

Avaliação preliminar de populações de cenoura para reação à mistura populacional de Meloidogyne incognita raça 1 e Meloidogyne javanica.

2009

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.

Non-nematode-derived double-stranded RNAs induce profound phenotypic changes in Meloidogyne incognita and Globodera pallida infective juveniles

Nine non-nematode-derived double-stranded RNAs (dsRNAs), designed for use as controls in RNA interference (RNAi) screens of neuropeptide targets, were found to induce aberrant phenotypes and an unexpected inhibitory effect on motility of root knot nematode Meloidogyne incognita J2s following 24 h soaks in 0.1 mg/ml dsRNA; a simple soaking procedure which we have found to elicit profound knockdown of neuronal targets in Globodera pallida J2s. We have established that this inhibitory phenomenon is both time- and concentration-dependent, as shorter 4 h soaks in 0.1 mg/ml dsRNA had no negative impact on M. incognita J2 stage worms, yet a 10-fold increase in concentration to 1 mg/ml for the same 4 h time period had an even greater qualitative and quantitative impact on worm phenotype and motility. Further, a 10-fold increase of J2s soaked in 0.1 mg/ml dsRNA did not significantly alter the observed phenotypic aberration, which suggests that dsRNA uptake of the soaked J2s is not saturated under these conditions. This phenomenon was not initially observed in potato cyst nematode G. pallida J2s, which displayed no aberrant phenotype, or diminution of migratory activity in response to the same 0.1 mg/ml dsRNA 24 h soaks. However, a 10-fold increase in dsRNA to 1 mg/ml was found to elicit comparable irregularity of phenotype and inhibition of motility in G. pallida, to that initially observed in M. incognita following a 24 h soak in 0.1 mg/ml dsRNA. Again, a 10-fold increase in the number of G. pallida J2s soaked in the same volume of 1 mg/ml dsRNA preparation did not significantly affect the observed phenotypic deviation. We do not observe any global impact on transcript abundance in either M. incognita or G. pallida J2s following 0.1 mg/ml dsRNA soaks, as revealed by reverse transcriptase-PCR and quantitative PCR data. This study aims to raise awareness of a phenomenon which we observe consistently and which we believe signifies a more expansive deficiency in our knowledge and understanding of the variables inherent to RNAi-based investigation.

Short interfering RNA-mediated gene silencing in Globodera pallida and Meloidogyne incognita infective stage juveniles

The analysis of gene function through RNA interference (RNAi)-based reverse genetics in plant parasitic nematodes (PPNs) remains inexplicably reliant on the use of long double-stranded RNA (dsRNA) silencing triggers; a practice inherently disadvantageous due to the introduction of superfluous dsRNA sequence. increasing chances of aberrant or off-target gene silencing through interactions between nascent short interfering RNAs (siRNAs) and non-cognate mRNA targets. Recently, we have shown that non-nematode, long dsRNAs have a propensity to elicit profound impacts on the phenotype and migrational abilities of both root knot and cyst nematodes. This study presents, to our knowledge for the first time, gene-specific knockdown of FMRFamide-like peptide (flp) transcripts, using discrete 21 bp siRNAs in potato cyst nematode Globodera pallida, and root knot nematode Meloidogyne incognita infective (J2) stage juveniles. Both knockdown at the transcript level through quantitative (q)PCR analysis and functional data derived from migration assay, indicate that siRNAs targeting certain areas of the FMRFamide-like peptide (FLP) transcripts are potent and specific in the silencing of gene function. In addition, we present a method of manipulating siRNA activity through the management of strand thermodynamics. Initial evaluation of strand thermodynamics as a determinant of RNA-induced Silencing Complex (RISC) strand selection (inferred from knockdown efficacy) in the siRNAs presented here suggested that the purported influence of 5' stand stability on guide incorporation may be somewhat promiscuous. However, we have found that on strategically incorporating base mismatches in the sense strand of a G. pallida-specific siRNA we could specifically increase or decrease the knockdown of its target (specific to the antisense strand), presumably through creating more favourable thermodynamic profiles for incorporation of either the sense (non-target-specific) or antisense (target-specific) strand into a cleavage-competent RISC. Whilst the efficacy of similar approaches to siRNA modification has been demonstrated in the context of Drosophila whole-cell lysate preparations and in mammalian cell cultures, it remained to be seen how these sense strand mismatches may impact on gene silencing in vivo, in relation to different targets and in different sequence contexts. This work presents the first application of such an approach in a whole organism; initial results show promise. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Evaluación de la actividad nematicida de cepas nativas y de colección de bacillus thuringiensis contra el nematodo formador de nódulos de la raíz (meloidogyne incognita).

Tesis (Doctor en Ciencias con especialidad en Biotecnología) UANL,2014.

Influence of pre-plant densities of Meloidogyne incognita on growth and root infestation of spinach (Spinacia oleracea L.) (Amaranthaceae) – an important dimension towards enhancing crop production

Vegetables represent a main source of micro-nutrients which can improve the health status of malnourished poor in the world. Spinach (Spinacia oleracea L.) is a popular leafy vegetable in many countries which is rich with several important micro-nutrients. Thus, consuming Spinach helps to overcome micro-nutrient deficiencies. Pests and pathogens act as major yield constraints in food production. Root-knot nematodes, Meloidogyne species, constitute a large group of highly destructive plant pests. Spinach is found to be highly susceptible for these nematode attacks. Though agricultural production has largely benefited from modern technologies and innovations, some important dimensions which can minimize the yield losses have been neglected by most of the growers. Pre-plant or initial nematode density in soil is a crucial biotic factor which is directly responsible for crop losses. Hence, information on preplant nematode densities and the corresponding damage is of vital importance to develop successful control procedures to enhance crop production. In the present study, effect of seven initial densities of M. incognita, i.e., 156, 312, 625, 1250, 2,500, 5,000 and 10,000 infective juveniles (IJs)/plant (equivalent to 1000cm3 soil) on the growth and root infestation on potted spinach plants was determined in a screen house. In order to ensure a high accuracy, root infestation was ascertained by the number of galls formed, the percentage galled-length of feeder roots and galled-feeder roots, and egg production, per plant. Fifty days post-inoculation, shoot length and weight, and root length were suppressed at the lowest IJs density. However, the pathogenic effect was pronounced at the highest density at which 43%, 46% and 45% reduction in shoot length and weight, and root length, respectively, was recorded. The highest reduction in root weight (26%) was detected at the second highest density. The Number of galls and percentage galled-length of feeder roots/per plant showed significant progressive increase across the increasing IJs density with the highest mean value of 432.3 and 54%, respectively. The two shoot growth parameters and root length showed significant inverse relationship with the increasing gall formation. Moreover, the shoot and root length were shown to be mutually dependent on each other. Suppression of shoot growth of spinach greatly affects the grower’s economy. Hence, control measures are essentially needed to ensure a better production of spinach via reducing the pre-plant density below the level of 0.156 IJs/cm3.

Resistência de clones de umezeiro e cultivares de pessegueiro a Meloidogyne incognita (nemata: Heteroderidae)

O presente estudo teve por objetivo avaliar a reação dos Clones 05; 10 e 15 de umezeiro (Prunus mume Sieb. et Zucc.) e das cultivares Okinawa, Aurora-1 e Dourado-1 de pessegueiro [Prunus persica (L.) Batsch] a Meloidogyne incognita (Kofoid and White) Chitwood, em condições de casa de vegetação. As plantas foram mantidas em vasos de cerâmica contendo uma mistura de solo e areia (1:1, v/v), previamente autoclavada a 121ºC e 1kgf.cm-2 por 2 horas. Aos 60 dias após o plantio, cada planta foi inoculada com 2.000 ovos e juvenis de segundo estádio de Meloidogyne incognita. O experimento foi conduzido em delineamento inteiramente casualizado, com 6 tratamentos (genótipos) e 9 repetições. Transcorridos 116 dias após a inoculação, as plantas foram colhidas para avaliação do sistema radicular. Foi possível verificar que o número de galhas por sistema radicular, o número de ovos e juvenis por 10g de raízes e por sistema radicular foi nulo ou praticamente nulo em todos os clones e nas cultivares estudadas, de forma que os respectivos fatores de reprodução foram todos inferiores a 1. Conclui-se que os Clones 05; 10 e 15 de umezeiro, assim como as cultivares Okinawa, Aurora-1 e Dourado-1 de pessegueiro são resistentes a Meloidogyne incognita.

Resistência de cultivares de batata-doce a Meloidogyne incognita

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Plantas antagonistas no manejo de Meloidogyne incognita, em solo arenoso de área de cultivo de olerícolas

The aim of this study was to evaluate the efficiency of antagonistic plants on nematode control in vegetables growing areas. The experiment was conducted in two periods in randomized complete block design in plots 1.5 x 1.4 m, corresponding to experimental units and randomly cultivated with the different plants. From each plot 100 cm(3) of soil and 10 g of tomato root were collected for estimating the initial population of the first and second experiment, respectively. Sixteen antagonistic plant seedlings of velvet bean (Stizolobium aterrimum), sunn plant (Crotalaria spectabilis) and pigeon pea (Cajanus cajan) were transferred to the plots and tomato (Solanun lycopersicum) cultivar Santa Clara was used as a control. After 116 days, two root systems and 100 cm(3) of soil were collected from each plot for a final nematode population analysis. Lettuce seedlings (Lactuca sativa) were transferred to the plots and evaluated after 28 and 42 days, respectively, for galls and eggs on the root system and fresh and dry weight of shoots,. Each treatment consisted of 6 replicates and the means were compared by LSD test (p<0.05). Meloidogyne incognita was found in the first survey. After the crop of the antagonistic plants, the M. incognita population in the root systems and the final population (soil + root) were statistically lower than in the control, which demonstrates the antagonistic effect of these plants on the nematode population. There were also a reduced number of galls on the lettuce cultivated after the antagonistic plants when compared to the control. The velvet bean and sunn plant showed an increase in dry shoot weight of the lettuce cultivated after the antagonists, respectively, in the first and second experiments.