Host-delivered RNAi: An effective strategy to silence genes in plant parasitic nematodes

Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

大功率微波与臭氧等复合因子对大棚蔬菜根结线虫及土壤的作

虫（nematodes）是世界农业生产的一大障碍，每年给世界农业生产造成约1000亿美元的巨额损失，是世界各国一直关注的重点病害之一。线虫可以危害各种大田作物和各种温室植物，几乎所有的栽培植物都有线虫危害发生。随着人们环保意识的增强以及健康意识的觉醒，传统防治根结线虫的药物如溴甲烷等因对环境的破坏作用，或者由于高毒、使用不便、抗药性等原因而迅速退出历史舞台，研究环保高效防治线虫成为世界各国竞相发展的技术之一。本研究尝试通过物理方式防治蔬菜根结线虫，并调查了对土壤质量的影响，主要内容包括： 1. 设计制造了功率20 Kw，频率为915 MHz的大功率土壤微波处理机，并通过了田间试验; 2. 设计制造了每小时120 g臭氧产量的便携式臭氧土壤处理机，并通过了田间试验; 3. 采用4因素3水平正交法L9(34)在山东寿光蔬菜大棚进行了蔬菜根结线虫的防治试验，综合考察微波、臭氧、微波吸收剂、EM菌等不同处理水平对根结线虫和对土壤质量的影响，结果表明： 1) 大棚土壤经微波60 s照射能够显著降低甜瓜根结线虫数和根结指数（p < 0.05），盆栽试验只需处理10 s即可显著防治甜瓜根结线虫（p < 0.01）;以120 g/h臭氧浓度处理5 s也能显著降低甜瓜根结线虫的数量（p < 0.05）;田间施入5 g/m2的EM菌，能够显著降低甜瓜根结线虫的数量（p < 0.05）;盆栽试验中微波吸收剂具有降低根结线虫数量的趋势，但是不同处理水平之间差异不显著，田间试验则具有增加根结线虫的趋势，不同处理水平之间也没有显著差异。 2) 微波不同处理水平之间对甜瓜单瓜重的影响没有显著差异;以120 g/h臭氧浓度处理甜瓜能够显著增加甜瓜单瓜重（p < 0.05）;200 g/m2微波吸收剂能够显著提高甜瓜单瓜重（p < 0.05）;以5 g/m2EM菌处理土壤则显著降低甜瓜单瓜重（p < 0.05）。 3) 微波、臭氧、微波吸收剂不同处理水平对甜瓜糖度的影响没有显著差异;以3 g/m2的EM菌处理甜瓜能够显著降低甜瓜的糖度。 4) 不同因素对甜瓜硬度的影响没有显著差异。 5) 微波处理60 s能够显著提高甜瓜的果型指数（p < 0.1），臭氧、微波吸收剂、EM菌等因素不同处理水平之间对果型指数影响差异不显著。 6) EM菌5 g/m2处理浓度能够显著降低甜瓜株高（p < 0.1），微波、臭氧、微波吸收剂不同处理水平之间对株高的影响没有显著差异。 7) EM菌5 g/m2处理浓度能够显著降低甜瓜根径（p < 0.1），微波、臭氧、微波吸收剂不同处理水平之间对根径的影响没有显著差异。 8) 微波处理60 s能够显著降低表层土壤（0-5 cm）的有机质含量（p < 0.01）;以120 g/h的臭氧浓度处理，则可以显著提高10-15 cm土壤的有机质含量（p < 0.05），其它处理因素的不同水平对有机质的影响差异不显著。 9) 微波不同处理水平对土壤全氮的影响没有显著差异（p < 0.05）;120 g/h臭氧处理浓度能够显著降低表层土壤（0-5 cm）的土壤全氮量（p < 0.1）;100 g/m2微波吸收剂处理水平能够显著提高10-15 cm土壤全氮量（p < 0.05）;EM菌3 g/m2的处理水平则显著降低5-10 cm土壤全氮量（p < 0.05）。 10) 微波、微波吸收剂、EM菌不同处理水平之间对土壤全磷没有显著影响;240 g/h臭氧浓度处理5 s能够显著降低10-15 cm土层的土壤全磷含量(p < 0.05)。 11) 微波、微波吸收剂对土壤全磷没有显著影响;臭氧处理具有降低土壤有效磷的趋势，EM菌处理则有提高土壤有效磷的趋势，但是 不同处理水平之间差异不显著（p > 0.05）。 12) 微波处理对土壤EC值没有显著影响;臭氧、微波吸收剂、EM菌处理具有降低土壤EC值的趋势，但是不同处理水平之间差异不显著（p > 0.05）。 13) 微波处理30 s能够显著降低表层土壤的pH值（p < 0.05），臭氧、微波吸收剂、EM菌处理具有提高土壤pH值的趋势，但是不同处理水平之间差异不显著。

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).

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.

Novel bioassays to examine the host-finding ability of plant-parasitic nematodes

We present two novel bioassays to be used in the examination of plant-parasitic nematode host-finding ability. The host-finding 'pipette-bulb assay' was constructed from modelled Pasteur pipette bulbs and connecting barrels using parafilm fastenings. This assay examines the direction of second-stage juvenile (J2) migration in response to a host seedling, through a moistened sand substrate, which underlies terminal upward-facing 'seedling bulbs', one containing a host seedling in potting compost, the other with only potting compost. An equal watering regime through both upward-facing seedling bulbs creates a directional concentration gradient of host diffusate chemotactic factors. Positive chemotactic stimuli cause the J2 to orientate and migrate towards the host plant. We present validation data collected from assays of the root-knot nematode, Meloidogyne incognita, and the potato cyst nematode, Globodera pallida, which indicate a highly significant positive attraction of J2 of both species to respective host plants. This represents a simple, quick and inexpensive method of assessing host-finding behaviour in the laboratory. We consider that the pipette-bulb assay improves on previous host-finding/chemo-attraction assays through creating a more biologically relevant environment for experimental J2; analysis is quick and easy, allowing the straightforward interpretation of results. In addition, we have developed an 'agar trough' sensory assay variant which we believe can be used rapidly to ratify nematode responses to chemical gustatory or olfactory cues. This was constructed from a water agar substrate such that two counting wells were connected by a raised central trough, all flooded with water. Two small water agar plugs were dehydrated briefly in an oven and then hydrated in either an attractant, repellent or water control; these plugs were then placed in the terminal counting wells and subsequently leached the attractant or repellent to form a concentration gradient along the central trough, which contained the initial J2 innoculum. Our data show that both M. incognita and G. pallida J2 are positively attracted to host diffusates. In addition, they displayed a strong repulsion in response to 1 M NaCl2. J2 of M. incognita displayed a mild aversion to a non-host oak root diffusate, whereas G. pallida J2 displayed a strong aversion to the same non-host diffusate; neither species responded to a compost leachate. We believe that the agar trough assay improves on previous methods by facilitating rapid diffusion of attractant or repellents. Both of the aforementioned assays were designed as tools to assess the impact of RNAi-based reverse genetics screens for gene targets involved in chemosensory orientation.

Biosecurity and Emerging Plant Health Problems in Turf Production and Maintenance

Recent research has supported the view that the distributions of many pests and diseases have extended towards higher latitudes over the last 50 years. Probably driven by a combination of climate change and trade, this extension to the ranges of hundreds of plant pathogens may have serious implications not only for agriculture, horticulture and forestry, but also for turf production &maintenance. Here we review our data relating to the current status of three emerging pest and disease problems across North West Europe (rapid blight, Labyrinthula sp. , the root knot nematode, Meloidogyne minor and the pacific stem gall nematode, Anguina pacificae ) and discuss the factors which may be involved in their spread and increasing impact on the turf industry. With turf production and maintenance becoming an increasingly international business, we ask if biosecurity and the promotion of plant health in turf production fields and associated sport facilities should be a greater priority for the industry. We also examine if a lack of effective biosecurity measures in the materials supply chain has led to increased plant health problems.

Evolução da população do fungo nematófago Pochonia chlamydosporia -isolado PcMR, após inoculação, no solo e na raiz do tomateiro cultivado em estufa

O fungo Pochonia chlamydospoia é um potencial agente de controlo biológico dos nemátodes-das-galhas-radiculares. Com este trabalho, pretendeu-se avaliar a eficácia de inoculações de clamidósporos no solo, no estabelecimento de uma população do fungo no solo e na raiz de tomateiro em estufa com níveis de densidade iguais ou superiores aos considerados como necessários para um eficaz controlo dos nematodes-das-galhas-radiculares. Ao longo de dois anos de ensaio, fórum efetuadas inoculações do isolado PcMR e avaliada a densidade de fungo no solo e na raiz, As inoculações efetuadas permitiram estabelecer uma população de P. chlamydosporia no solo e atingir os valores de densidade pretendidos. No entanto, os valores pretendidos para colonização da raiz pelo fungo foram atingidos apenas no primeiro ano. Foi igualmente demonstrada a capacidade do fungo em se manter no solo durante longos períodos de tempo mesmo na ausência de cultura e em condições adversas de humidade e temperatura. /ABSTRACT: Pochonia chlamydosporia is a potential root-knot nematode biological control agent. The aim of this work was to evaluate the effectiveness of chlamydospore inoculations at the soil, for the establishment at both soil and greenhouse tomato root, of a fungus population in density levels equal or superior to those considered as needed for an effective control of root-knot nematode. Along two years, several inoculations using the Portuguese isolate PcMR were made and the density of fungus at the soil and roots studied. These inoculations allowed the establishment of a population of P. chlamydosporia at the soil and achieve the desired density values. However, only in the first year of assay, the desired values of root colonization by fungus were achieved. lt was also demonstrated that P. chlamydosporia can survive for itself at the soil for a long period of time even in the absence of plant culture and in adverse moist and temperature conditions.

Characterization and efficacy of bacterial strains for biological control of soil-borne diseases caused by Phytophthora cactorum and Meloidogyne javanica on Rosaceous plants

S'avaluaren 58 soques de Pseudomonas fluorescens i Pantoea agglomerans per la seva eficàcia en el biocontrol de la malaltia causada per l'oomicet Phytophthora cactorum en maduixera i pel nematode formador de gal·les Meloidogyne javanica en el portaempelt GF-677. Es desenvolupà un mètode ex vivo d'inoculació de fulla amb l'objectiu de seleccionar soques bacterianes com a agents de control biològic de P. cactorum en maduixera. Tres soques de P. fluorescens es seleccionaren com a soques eficaces en el biocontrol del patogen en fulles i en la reducció de la malaltia en plantes de maduixera. La combinació de soques semblà millorar la consistència del biocontrol en comparació amb les soques aplicades individualment. Tres soques de P. fluorescens es seleccionaren per la seva eficàcia en la reducció de la infecció de M. javanica en portaempelts GF-677. La combinació d'aquestes soques no incrementà l'eficàcia del biocontrol, però semblà reduir la seva variabilitat.

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax). (c) 2007 Elsevier Inc. All rights reserved.

The isolation of a single spore isolate of Pasteuria penetrans and its pathogenicity on Meloidogyne javanica

We have obtained a single spore isolate of Pasteuria penetrans, derived by allowing a single spore to attach to a second-stage juvenile (J2) of the root-knot nematode Meloidogyne javanica. By analysing DNA sequences at three different loci we have obtained evidence that the isolate is, indeed, genetically pure. We compared the ability of the single spore isolate and the parent population from which it was selected to attach to and parasitise both the original population of M. javanica on which it was isolated and a single egg mass line derived from it. There was no difference in the attachment of spores of the single spore isolate to juveniles compared to the parental population, although there were higher numbers of both attaching to J2 of the single egg mass line compared to its parental population. Judging from the numbers of egg masses and Pasteuria-infected females, the single spore isolate was less pathogenic to the parental population of M. javanica than was the parental spore population.

Levantamento de Meloidogyne exigua na cultura da seringueira em São José do Rio Claro, MT, Brasil

Este trabalho foi realizado com o objetivo de efetuar um levantamento da ocorrência de Meloidogyne exigua em seringueira em São José do Rio Claro, MT. Foram amostradas 191 propriedades agrícolas, totalizando cerca de 18.000ha. Os nematóides foram identificados no Laboratório de Nematologia do Departamento de Fitossanidade da FCAV/UNESP, em Jaboticabal, SP. Foram encontrados níveis populacionais de M. exigua entre 0 e 61.824 juvenis/5g de raízes.

Ocorrência de Meloidogyne enterolobii em muricizeiro (Byrsonima cydoniifolia)

A entrada de agentes fitopatogênicos em novas localidades através de mudas infectadas constitui uma das principais formas de disseminação. Meloidogyne enterolobii é uma espécie de nematoide altamente virulenta que tem causado sérios danos a plantas cultivadas no Brasil. Neste trabalho é relatada a primeira ocorrência de M. enterolobii em mudas de muricizeiro (Byrsonima cydoniifolia), uma planta nativa da Amazônia e em mudas de goiabeira (Psidium guajava), no Estado de Mato Grosso. Com base nos caracteres morfológicos do padrão perineal de fêmeas, região labial dos machos e no fenótipo isoenzimático de esterase, foi confirmado que a espécie encontrada tanto nas mudas de muricizeiro quanto nas de goiabeira é M. enterolobii.

Reação de clones de umezeiro (Prunus mume sieb. et zucc.) e cultivares de pessegueiro a Meloidogyne javanica (treub, 1885) Chitwood, 1949

Um amplo projeto de estudos sobre a utilização do umezeiro como porta-enxerto para pessegueiro está sendo desenvolvido na FCAV/UNESP, Câmpus de Jaboticabal-SP, devido, especialmente, às promissoras características para uso como redutor de vigor da copa e sua boa qualidade de frutos. Alguns trabalhos na literatura citam o umezeiro como resistente ao nematóide das galhas, entretanto dispõe-se de poucas informações. Neste trabalho, teve-se por objetivo estudar a reação de clones de umezeiro e cultivares de pessegueiro a Meloidogyne javanica. O experimento foi conduzido em casa de vegetação, com 6 tratamentos (Clones 05; 10 e 15 de umezeiro e as cultivares Okinawa, Aurora-1 e Dourado-1 de pessegueiro) e 9 repetições. 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 sessenta dias após o plantio, cada planta foi inoculada com 3.000 ovos e juvenis de segundo estádio de Meloidogyne javanica. Aos 100 dias após a inoculação, as plantas foram colhidas para avaliação da massa de matéria fresca do sistema radicular, número de galhas por sistema radicular, número de ovos e juvenis por 10 g de raízes, número de ovos e juvenis por sistema radicular e fator de reprodução. Verificou-se que todos os clones e cultivares de umezeiro e pessegueiro, respectivamente, mostraram-se resistentes a Meloidogyne javanica.

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.