Activity profiling of barley vacuolar processing enzymes provides new insights into the plant and cyst nematode interaction

Vacuolar processing enzymes (VPEs) play an important role during regular growth and development and defence responses. Despite substantial attempts to understand the molecular basis of plant–cyst nematode interaction, the mechanism of VPEs functioning during this interaction remains unknown. The second-stage Heterodera filipjevi juvenile penetrates host roots and induces the formation of a permanent feeding site called a syncytium. To investigate whether infection with H. filipjevi alters plant host VPEs, the studies were performed in Hordeum vulgare roots and leaves on the day of inoculation and at 7, 14 and 21 days post-inoculation (dpi). Implementing molecular, biochemical and microscopic methods we identified reasons for modulation of barley VPE activity during interaction with H. filipjevi. Heterodera filipjevi parasitism caused a general decrease of VPE activity in infected roots, but live imaging of VPEs showed that their activity is up-regulated in syncytia at 7 and 14 dpi and down-regulated at 21 dpi. These findings were accompanied by tissue-specific VPE gene expression patterns. Expression of the barley cystatin HvCPI-4 gene was stimulated in leaves but diminished in roots upon infestation. External application of cyclotides that can be produced naturally by VPEs elicits in pre-parasitic juveniles vesiculation of their body, enhanced formation of granules, induction of exploratory behaviour (stylet thrusts and head movements), production of reactive oxygen species (ROS) and final death by methuosis. Taken together, down-regulation of VPE activity through nematode effectors promotes the nematode invasion rates and leads to avoidance of the induction of the plant proteolytic response and death of the invading juveniles.     

An unconventionally secreted effector from the root knot nematode Meloidogyne incognita,Mi-ISC-1, promotes parasitism by disrupting salicylic acid biosynthesis in host plants

Plant-parasitic nematodes need to deliver effectors that suppress host immunity for successful parasitism. We have characterized a novel isochorismatase effector from the root-knot nematode Meloidogyne incognita, named Mi-ISC-1. The Mi-isc-1 gene is expressed in the subventral oesophageal glands and is up-regulated in parasitic-stage juveniles. Tobacco rattle virus-induced gene silencing targeting Mi-isc-1 attenuated M. incognita parasitism. Enzyme activity assays confirmed that Mi-ISC-1 can catalyse hydrolysis of isochorismate into 2,3-dihydro-2,3-dihydroxybenzoate in vitro. Although Mi-ISC-1 lacks a classical signal peptide for secretion at its N-terminus, a yeast invertase secretion assay showed that this protein can be secreted from eukaryotic cells. However, the subcellular localization and plasmolysis assay revealed that the unconventional secretory signal present on the Mi-ISC-1 is not recognized by the plant secretory pathway and that the effector was localized within the cytoplasm of plant cells, but not apoplast, when transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Ectopic expression of Mi-ISC-1 in N. benthamiana reduced expression of the PR1 gene and levels of salicylic acid (SA), and promoted infection by Phytophthora capsici. The cytoplasmic localization of Mi-ISC-1 is required for its function. Moreover, Mi-ISC-1 suppresses the production of SA following the reconstitution of the de novo SA biosynthesis via the isochorismate pathway in the cytoplasm of N. benthamiana leaves. These results demonstrate that M. incognita deploys a functional isochorismatase that suppresses SA-mediated plant defences by disrupting the isochorismate synthase pathway for SA biosynthesis to promote parasitism.     

Muscodor albus, a potential biocontrol agent against plant-parasitic nematodes of economically important vegetable crops in Washington State, USA

The fungus, Muscodor albus, was tested for nematicidal and nematostatic potential against four plant-parasitic nematode species with three different feeding modes on economically important vegetable crops in the Pacific Northwest. Meloidogyne chitwoodi, Meloidogyne hapla, Paratrichodorus allius, and Pratylenchus penetrans were exposed for 72 h to volatiles generated by M. albus cultured on rye grain in sealed chambers at 24 °C in the laboratory. In addition, the nematodes were inoculated into soil fumigated with M. albus, and incubated for 7 days prior to the introduction of host plants under greenhouse conditions. The mean percent mortality of nematodes exposed to M. albus in the chamber was 82.7% for P. allius, 82.1% for P. penetrans, and 95% for M. chitwoodi; mortality in the nontreated controls was 5.8%, 7%, and 3.9%, respectively. Only 21.6% of M. hapla juveniles died in comparison to 8.9% in controls. However, 69.5% of the treated juveniles displayed reduced motility and lower response to physical stimulus by probing, in comparison to the control juveniles. This is evidence of nematostasis due to M. albus exposure. The greenhouse study showed that M. albus caused significant reduction to all nematode species in host roots and in rhizosphere soil. The percent mortality caused by M. albus applied at 0.5% and 1.0% w/w in comparison to the controls was as follows: 91% and 100% for P. allius in the soil; 100% for P. penetrans in bean roots and soil; 85% and 95% for M. chitwoodi in potato roots, and 56% and 100% in the soil; 100% for M. hapla both in pepper roots and soil. In this study, M. albus has shown both nematostatic and nematicidal properties.     

Two Simple Methods for the Collection of Individual Life Stages of Reniform Nematode,Rotylenchulus reniformis

The sedentary semi-endoparasitic nematode Rotylenchulus reniformis, the reniform nematode, is a serious pest of cotton and soybean in the United States. In recent years, interest in the molecular biology of the interaction between R. reniformis and its plant hosts has increased; however, the unusual life cycle of R. reniformis presents a unique set of challenges to researchers who wish to study the developmental expression of a particular nematode gene or evaluate life stage–specific effects of a specific treatment such as RNA-interference or a potential nematicide. In this report, we describe a simple method to collect R. reniformis juvenile and vermiform adult life stages under in vitro conditions and a second method to collect viable parasitic sedentary females from host plant roots. Rotylenchulus reniformis eggs were hatched over a Baermann funnel and the resultant second-stage juveniles incubated in petri plates containing sterile water at 30°C. Nematode development was monitored through the appearance of fourth-stage juveniles and specific time-points at which each developmental stage predominated were determined. Viable parasitic sedentary females were collected from infected roots using a second method that combined blending, sieving, and sucrose flotation. Rotylenchulus reniformis life stages collected with these methods can be used for nucleic acid or protein extraction or other experimental purposes that rely on life stage–specific data.     

A novel Meloidogyne graminicola effector,MgMO237, interacts with multiple host defence‐related proteins to manipulate plant basal immunity and promote parasitism

Plant‐parasitic nematodes can secrete effector proteins into the host tissue to facilitate their parasitism. In this study, we report a novel effector protein, MgMO237, from Meloidogyne graminicola, which is exclusively expressed within the dorsal oesophageal gland cell and markedly up‐regulated in parasitic third‐/fourth‐stage juveniles of M. graminicola. Transient expression of MgMO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cells. Rice plants overexpressing MgMO237 showed an increased susceptibility to M. graminicola. In contrast, rice plants expressing RNA interference vectors targeting MgMO237 showed an increased resistance to M. graminicola. In addition, yeast two‐hybrid and co‐immunoprecipitation assays showed that MgMO237 interacted specifically with three rice endogenous proteins, i.e. 1,3‐β‐glucan synthase component (OsGSC), cysteine‐rich repeat secretory protein 55 (OsCRRSP55) and pathogenesis‐related BetvI family protein (OsBetvI), which are all related to host defences. Moreover, MgMO237 can suppress host defence responses, including the expression of host defence‐related genes, cell wall callose deposition and the burst of reactive oxygen species. These results demonstrate that the effector MgMO237 probably promotes the parasitism of M. graminicola by interacting with multiple host defence‐related proteins and suppressing plant basal immunity in the later parasitic stages of nematodes.     

A possible new approach for controlling root knot nematode,Meloidogyne incognita,infecting eggplant by certain insect growth regulators

Under greenhouse conditions, three insect growth regulators, buprofezin, flufenoxuron and pyriproxyfen, at field concentrations and other two lower concentrations were evaluated for controlling of root knot nematode, Meloidogyne incognita, infecting eggplant (Solanum melongena L.) cv. Baladi. Generally, the tested pesticides reduced the number of galls, egg masses, females and developmental stages in roots and juveniles in soil compared to untreated check. The percentages of reduction were positively proportional with the respective concentrations. Flufenoxuron at its highest concentration was the most effective pesticide in reducing galls, egg masses, females and developmental stages compared with pyriproxyfen and buprofezin. The percentages of reduction caused by flufenoxuron were 67.80, 69.57, 70.75 and 95.23% for the, respective nematode criteria, while buprofezin at the highest concentration was the most effective in reducing number of hatched juveniles in soil followed by flufenoxuron and pyriproxyfen. Data showed that the length and fresh and dry weights of root and shoot of egg plant were increased with the lowest concentrations than the highest ones.     

Nematode–citrus plant interactions: host preference,damage rate and molecular characterization of Citrus root nematode Tylenchulus semipenetrans

• Citrus plants are host to several plant parasitic nematodes (PPNs), which are microscopic organisms. Among PPNs, the citrus root nematode, T. semipenetrans (Cobb 1913) (Tylenchida: Tylenchulidae), causes significant damage to citrus plantations worldwide. Understanding citrus nematode populations, precise identification, host preference among citrus species, and damage threshold are crucial to control T. semipenetrans. The minutiae of citrus plant–nematode interactions, nematode density and molecular nematode identification are not well understood. In this study, nematode species and density in citrus orchards, host specialization, molecular and morphological characteristics of nematodes were assessed.
• Molecular and morphological methods, host–nematode interactions, host (citrus species) preference, damage economic threshold (ET), and economic injury level (EIL) were determined using laboratory methods and field sampling. Citrus plantations in different provinces in the Mediterranean region of Turkey were investigated.
• Nematode species were identified molecularly and morphologically. ITS sequences revealed that samples were infected by citrus root nematode T. semipenetrans. The lowest nematode density was in C. reticulata in Mersin (53 2nd stage juveniles (J2s) 100 g⁻¹ soil), while the highest density was from Hatay in C. sinensis (12173 J2s 100 g⁻¹ soil). Highest citrus nematode population density was on roots of C. reticulata, followed by C. sinensis, C. limon, and C. paradisi.
• The citrus nematode is more common than was thought and population fluctuations change according to specific citrus species. Environmental conditions, host and ecological factors, such as temperature, soil pH, and soil nutrients, might influence nematode populations in citrus orchards. Investigating nematode density in diverse soil ecologies and the responses of different resistant/tolerant citrus species and cultivars to nematode populations is essential in future studies.

     

Behaviour of the systemic nematicide oxamyl in plants in relation to control of invasion and development of Meloidogyne incognita

A single foliar application of oxamyl (12.5 μg) in acetone significantly reduced invasion of cucumber seedlings by Meloidogyne incognita juveniles for at least 21 days but did not affect the early stages of development of juveniles which had already invaded the roots. In contrast, application of oxamyl to the roots significantly reduced both invasion and development of juveniles. Concurrent studies using radiolabelled oxamyl showed that the amount of toxicant in the roots after 3 days was 13 times greater following root application than after foliar treatment. It is probable that oxamyl concentrates at the sites of nematode attack as an overall concentration of only 3 ng oxamyl g^-1 root was sufficient to prevent invasion. Much greater concentrations than this were required to affect the nematode in vitro. Oxamyl appeared to be lost from the roots into the soil principally in the form of its non-toxic oxime and it is suggested that the site of action following foliar application is at the root surface or outer cortex. Studies on the invasion behaviour of M. incognita juveniles on agar showed that the action of oxamyl had a sensory component.     

Hot water immersion of Allium hookeri roots for the control of the plant parasitic nematodes Meloidogyne javanica and Pratylenchus coffeae

Nematodes are important quarantine pests of bulbous plants such as hooker chives. Although control methods such as fumigation, chemical immersion, and heat are often applied, it has proved difficult to disinfect nematodes from plant roots in quarantine. As heat treatment has been successfully useful for the control of nematodes in other agricultural products in quarantine, we investigated the susceptibility and mortality rates of Meloidogyne javanica and Pratylenchus coffeae, which infest hooker chive roots, using a hot water immersion method. Heat damage to the hooker chive roots was noticeable at temperatures over 50°C. Temperatures for the effective time to kill 99% at 1 min (ET99) for M. javanica and P. coffeae juveniles were 49.3°C and 49.1°C, respectively. However, the time to kill 99% of M. javanica eggs at 48°C and 49°C were 27.0 min and 8.3 min, respectively. Using a thermal equilibrium formula, the optimum commercial scale condition, in a 1400‐L chamber, for nematode control without associated plant damage was water immersion at 48.2°C for 30 min or at 49.2°C for 13 min with a filling ratio less than 12%. This result can be applicable for the nematode disinfestation of hooker chive roots in plant quarantine.     

Antagonistic Potential of Bacillus pumilus L1 Against Root‐Knot Nematode,Meloidogyne arenaria

This study was conducted to estimate the potential of Bacillus pumilus L1 against root‐knot nematode, Meloidogyne arenaria, in both in vitro and in vivo conditions. B. pumilus L1 was found to produce both protease and chitinase. When various concentrations (1–10%) of the bacterial culture (BC) or 0.02–0.11 mg/ml of the crude enzymes produced by B. pumilus L1 were used to treat M. arenaria eggs and second‐stage juveniles (J2), inhibition of hatching and J2 mortality were significantly increased under in vitro conditions. In addition, the hatching inhibition and J2 mortality rate were improved with increasing concentrations of BC and the crude enzymes. Similarly, these effects also increased over time after treatment with BC. Moreover, the crude enzymes caused partial degradation of the eggshell and juvenile body when treated at 0.11 mg/ml. The pot experiment also demonstrated that the application of BC to potted soil caused significant reduction in the number of galls and egg masses in the plant roots and of the J2 population as compared to the untreated control 6 weeks after M. arenaria infestation. In addition, the simultaneous application of BC upon nematode inoculation proved more effective than application 2 days postinoculation with nematode. B. pumilus L1 inoculation (BC, BCs and BC2) also promoted tomato plant growth as compared to the controls (TW, Ne, GM and NeT). Thus, our results demonstrated the ability of B. pumilus L1 as a potential biocontrol agent against root‐knot nematode, with additional activity as a plant growth promoter for tomato.     

Arbuscular mycorrhizal fungi affect both penetration and further life stage development of root-knot nematodes in tomato

The root-knot nematode Meloidogyne incognita poses a worldwide threat to agriculture, with an increasing demand for alternative control options since most common nematicides are being withdrawn due to environmental concerns. The biocontrol potential of arbuscular mycorrhizal fungi (AMF) against plant-parasitic nematodes has been demonstrated, but the modes of action remain to be unraveled. In this study, M. incognita penetration of second-stage juveniles at 4, 8 and 12 days after inoculation was compared in tomato roots (Solanum lycopersicum cv. Marmande) pre-colonized or not by the AMF Glomus mosseae. Further life stage development of the juveniles was also observed in both control and mycorrhizal roots at 12 days, 3 weeks and 4 weeks after inoculation by means of acid fuchsin staining. Penetration was significantly lower in mycorrhizal roots, with a reduction up to 32%. Significantly lower numbers of third- and fourth-stage juveniles and females accumulated in mycorrhizal roots, at a slower rate than in control roots. The results show for the first time that G. mosseae continuously suppresses root-knot nematodes throughout their entire early infection phase of root penetration and subsequent life stage development.     

Interaction of the Microbivorous Nematode Teratorhabditis dentifera and the Nematode-Pathogenic Fungus Hirsutella rhossiliensis

The fungus Hirsutella rhossiliensis is an obligate pathogen with a broad host range among nematodes. Microbivorous nematodes are abundant around plant roots and may serve as hosts for the fungus. Our objective was to determine the influence of the bacterial-feeding nematode Teratorhabditis dentifera on the abundance of H. rhossiliensis. Experiments were conducted in a growth chamber with pots containing pasteurized soil, the fungus, and potato plants. The abundance of infectious conidia was compared in pots with and without T. dentifera after 50 or 70 days. The nematode reached high densities (10-40/cm³ soil) but had no effect on the abundance of conidia. Many individuals were dauer juveniles, a stage that acquired conidia but did not become infected. To test whether this life stage could deplete the pool of conidia in soil, different proportions of dauer juveniles with (resistant) and without (susceptible) a sheath were added to H. rhossiliensis-infested soil. The number of conidia in the soil decreased with an increasing proportion of resistant nematodes. Different stages of T. dentifera appear to have opposing effects on H. rhossiliensis; while adults and regular juveniles acquire conidia, become infected, and produce new infectious conidia, dauer juveniles can deplete the supply of conidia.     

Multifaceted Strategies Used by Root-Knot Nematodes to Parasitize Plants-A Review

Root-knot nematodes being omnipresent in agricultural and horticultural soils are tallied among the most important economic pathogens around the world. For successful parasitism, these nematodes use various strategies to control and manipulate the host plant’s cell machinery. These strategies include the molecular mimicry of some host genes by some nematode secreted effector proteins, secretion of cell wall digesting enzymes and other effector proteins that are responsible for the suppression of defence by the host plant. All these secretions which are released through the stylet, contribute to the formation of specialized feeding sites or giant cells. The effector proteins interfere with the normal physiology, cytology and biochemistry of the host plant. The present review brings novel insights by summarizing some novel effectors that have been discovered recently like MgPDI, MiMIF, MiIDL1, MiISE6, Mg16820, etc. It also discusses some novel mechanisms through which these effector proteins target different pathways of host plants and thus facilitate nematode parasitism.     

Histopathology of Okra and Ridgeseed Spurge Infected with Meloidodera charis

Histological observations of okra Abelomoschus esculentus ''Clemson Spineless'' and ridgeseed spurge Euphorbia glyptosperma (a common weed) infected with Meloidodera charis Hopper, indicated that the juvenile nematode penetrated the roots intercellularly. Within 5 days after plant emergence the nematode positioned its body in the cortical tissue parallel to the vascular system. By 10 days after plant emergence the juvenile had extended its head into the vascular system and initiated giant cell formation, generally in protophloem tissue. Giant cells were one celled and usually multi-nucleate. Eggs were observed in the female body 30 days after plants emerged and juveniles were found within the female body by 40 days. Nematode development progressed equally in the root system of either host plant. Generally, throughout the nematode''s life cycle its entire body remained inside the cortical tissue of okra. In ridgeseed spurge, however, the posterior portion of the female erupted through the host epidermis as early as 15 days after plant emergence; only the head and neck remained embedded in the host. The nematode caused extensive tissue disruption in the cortical and vascular system of both plant species. Corn, Zea mays, was another host of the nematode.     

Perception and Signal Transduction of Rhizobial NOD Factors

Referee: Dr. Gary Stacey, Director, Center for Legume Research, Department of Microbiology, M409 Walters Life Science Bldg., University of Tennessee, Knoxville, TN 37966-0845 Soil bacteria belonging to genera Rhizobium, Bradyrhizobium, Allorhizobium, Azorhizobium, Mesorhizobium, and Sinorhizobium are able to induce nodule formation on the roots of leguminous plants. In the differentiated root nodules bacteria fix as bacteroids atmospheric nitrogen and deliver it to the host plant. The interaction between bacteria and host plant starts with a complex signal exchange. After induction by plant flavonoids, rhizobia synthesize and secrete lipo-chitooligosaccharides (LCOs), known as Nod factors, which induce morphological changes and expression of early nodulin genes in the roots of host plants. Specific recognition of Nod factors by host plants and early stages of signal transduction are discussed.     

Arabidopsis HIPP27 is a host susceptibility gene for the beet cyst nematode Heterodera schachtii

Sedentary plant‐parasitic cyst nematodes are obligate biotrophs that infect the roots of their host plant. Their parasitism is based on the modification of root cells to form a hypermetabolic syncytium from which the nematodes draw their nutrients. The aim of this study was to identify nematode susceptibility genes in Arabidopsis thaliana and to characterize their roles in supporting the parasitism of Heterodera schachtii. By selecting genes that were most strongly upregulated in response to cyst nematode infection, we identified HIPP27 (HEAVY METAL‐ASSOCIATED ISOPRENYLATED PLANT PROTEIN 27) as a host susceptibility factor required for beet cyst nematode infection and development. Detailed expression analysis revealed that HIPP27 is a cytoplasmic protein and that HIPP27 is strongly expressed in leaves, young roots and nematode‐induced syncytia. Loss‐of‐function Arabidopsis hipp27 mutants exhibited severely reduced susceptibility to H. schachtii and abnormal starch accumulation in syncytial and peridermal plastids. Our results suggest that HIPP27 is a susceptibility gene in Arabidopsis whose loss of function reduces plant susceptibility to cyst nematode infection without increasing the susceptibility to other pathogens or negatively affecting the plant phenotype.