Effective delivery of a nematode‐repellent peptide using a root‐cap‐specific promoter

The potential of the MDK4‐20 promoter of Arabidopsis thaliana to direct effective transgenic expression of a secreted nematode‐repellent peptide was investigated. Its expression pattern was studied in both transgenic Arabidopsis and Solanum tuberosum (potato) plants. It directed root‐specific β‐glucuronidase expression in both species that was chiefly localized to cells of the root cap. Use of the fluorescent timer protein dsRED‐E5 established that the MDK4‐20 promoter remains active for longer than the commonly used constitutive promoter CaMV35S in separated potato root border cells. Transgenic Arabidopsis lines that expressed the nematode‐repellent peptide under the control of either AtMDK4‐20 or CaMV35S reduced the establishment of the beet cyst nematode Heterodera schachtii. The best line using the AtMDK4‐20 promoter displayed a level of resistance >80%, comparable to that of lines using the CaMV35S promoter. In transgenic potato plants, 94.9 ± 0.8% resistance to the potato cyst nematode Globodera pallida was achieved using the AtMDK4‐20 promoter, compared with 34.4 ± 8.4% resistance displayed by a line expressing the repellent peptide from the CaMV35S promoter. These results establish the potential of the AtMDK4‐20 promoter to limit expression of a repellent peptide whilst maintaining or even improving the efficacy of the cyst‐nematode defence.     

Comparison of damage levels caused by Radopholus similis and Helicotylenchus multicinctus on bananas in Uganda

Field surveys were done in 1997 and 1998 in Masaka district, Uganda, an area which is experiencing a decline in banana production, to assess level of damage caused by nematodes Radopholus similis and Helicotylenchus multicinctus at farm level. Ten farms within a radius of 2 km were selected and nematode damage assessed. The major nematodes encountered were Pratylenchus goodeyi, R. similis and H. multicinctus. P. goodeyi and H. multicinctus were more abundant than R. similis but R. similis had a stronger and significant correlation with root damage. A subsequent pot trial examined pathogenicities of R. similis and H. multicinctus in pure and mixed cultures on tissue cultured‐banana plantlets. R. similis alone and in mixed population reduced root fresh weight significantly, and the percentage of root necrosis ranged between 22.8–41.6% and 18.3–45.5% for March 1998 and March 1999 trials, respectively. The difference in damage caused by R. similis alone and in mixed population was not statistically significant, and was higher than the damage caused by H. multicinctus alone. There were no significant differences in pathogenicity among the R. similis isolates from different parts of Uganda.     

Relationships between nematodes, weevils, banana and plantain cultivars and damage

Infestation of banana and plantain suckers by nematodes (Pratylenchus good-eyi, Radopholus similis and Helicotylenchus multicinctus) and weevils (Cosmopolites sordidus) increased with time from establishment in a field of eight different cultivars. There was a strong association between nematode and weevil infestation; suckers infested with nematodes were more than four times more likely to be infected by weevil than suckers without nematodes. Weevil damage, measured by percentage coefficient of infestation (PCI) at harvest of the bunch, was higher on a plantain (cv. Gonja) and on an east African highland cooking banana (cv. Lusumba) than on the sweet and multi-purpose cultivars. There was a correlation between numbers of male weevils caught in a plot and the mean PCI measured in the plot, but no correlation with female numbers.     

Phytopathological strategies to combat some diseases of plantain for improved nutrition,income generation and poverty alleviation

The problem of micronutrient deficiencies in the diet of indigenous people of Imo State can be solved not only by improving the diet, but also by identifying nutritious foods as well as making such foods available both in quantities and qualities that needed to improve health. Plantain is widely consumed in many ways such as baked, fried, boiled, steamed, dried or eaten raw and the management demand for plantain make production expensive and out of the reach of resource for poor indigenes. Plantain production must cope with numerous environmental challenges particularly with fungal, bacteria, viral and nematode pathogens as well as pests and non-pathogenic diseases which reduce plantain production. Plantain is highly rich in iron, zinc and β-carotene whose deficiencies are commonly reported in children and women of reproductive age. The most serious disease of plantain is the black Sigatoka caused by Mycosphaerella fijiensis. Others include Fusarium wilt, anthracnose, bacteria wilt, fruit rot, burrowing nematode Radopholus similis and bract mosaic. There had been scientific approaches to combat these diseases such as use of chemicals, biological enhancement, crop sanitation, use of alternative and collateral hosts as well as genetic manipulations.     

Expression of a plant cystatin confers partial resistance to Globodera,full resistance is achieved by pyramiding a cystatin with natural resistance

Constructs based on a cysteine proteinase inhibitor (cystatin) from sunflower and a protein engineered variant of a rice cystatin (Oc-ID86) conferred similar levels of resistance on potato plants to Globodera spp as chicken egg white cystatin (CEWC) under the control of CaMV35S. The level of resistance on challenge by 6.5 viable eggs Globodera spp g^–1 soil in a small scale field trial was similar to that provided by the natural partial resistance of cv. Sante. PCR of an internal transcribed spacer region of the ribosomal cistron of the nematode genome established that the challenging population was a mixture of G. pallida and G. rostochiensis with no evidence of a differential effect of the transgenic resistance on the reproductive success of the two species. Transformation of Sante and the South American cultivar Maria Huanca with CaMV35S/CEWC raised the status of both cultivars from partial to full resistance in this study. The results establish the potential of plant cystatins and demonstrate an additive effect of expressing CEWC with natural resistance genes.     

The development of plant-parasitic nematode infestations on micro-propagated banana plants following field control measures in Côte d'Ivoire

Of the weeds that were found in banana production areas, only Asystasia gangetica was parasitised by Radopholus similis; Helicotylenchus multicinctus and Hoplolaimus pararobustus were able to parasitise all weed species. Field trials were carried out in Cote d'Ivoire to assess the potential for using nematode-free micro-propagated banana plants following cultural and chemical methods for nematode control. Banana (Musa acuminata) cv. Poyo was examined for nematodes after weed fallow, flooding and chemical treatment. Before replanting bananas, nematode assays showed that: i) all nematode species declined but were not eliminated after a 1,3-dichloropropene soil treatment; ii) H. multicinctus, H. pararobustus and Cephalenchus emarginatus were still present after either a 2-year weed fallow (dominated by Chromolaena odorata or Asystasia gangetica) or a 10-week flooding; iii) R. similis did not persist after fallowing or flooding. All nematode species were found in plots treated with 1,3-dichloropropene and that had been planted with bullheads, suckers or nematode-free micropropagated plants. After both fallow and flooding, R. similis was reintroduced on infested planting materials (bullheads and suckers) even when they were pre-treated with a nematicide. When fallow and flooded plots were planted with nematode-free materials (vitro-plants), R. similis did not appear in the roots for two vegetative cycles. The other species, still present in the soil, invaded the roots and increased slowly in numbers. These results were confirmed in commercial banana plantations.     

Banana Rhizodeposition: Characterization of Root Border Cell Production and Effects on Chemotaxis and Motility of the Parasitic Nematode Radopholus similis

Rhizodeposition was collected from root tips of banana (Musa acuminata). Two varieties, Grande naine and Yangambi km5, susceptible and resistant towards the burrowing nematode Radopholus similis, respectively, were examined for root border cell production under in vitro and in soil growing conditions. Two types of cells were observed in rhizodeposition: spherical cells containing large amyloplasts, called statocytes (8% of total), and long ellipsoidal border cells (92%). Border cell production was high, related to root length and not different between the two cultivars. Rhizodeposition from plants grown in soil contained similar amounts of border cells, but viability was lower than in in vitro grown plants. Chemotaxis and motility assays were performed to test the effects of banana roots, roots without rhizodeposition, rhizodeposition, exudates and border cells on the behaviour of R. similis. Roots of both the susceptible and resistant variety attracted R. similis, but only in the presence of rhizodeposition. Isolated rhizodeposition of Yangambi km5 also attracted nematodes. Border cells and exudates did not affect nematode chemotaxis. Roots of both varieties induced quiescence in R. similis in the chemotaxis assay. Thirty to fifty percent of the nematodes became temporary quiescent in the motility assay with rhizodeposition and exudates. The effect lasted for 24 h for Grande naine and up to 3 days for Yangambi km5. Rhizodeposition collected from plants grown in soil affected neither R. similis chemotaxis nor motility. Overall, there were no indications that rhizodeposition is involved in preformed resistance against R. similis in Yangambi km5.     

Quantitative Digital Imaging of Banana Growth Suppression by Plant Parasitic Nematodes

A digital camera fitted with a hemispherical lens was used to generate canopy leaf area index (LAI) values for a banana (Musa spp.) field trial with the aim of establishing a method for monitoring stresses on tall crop plants. The trial in Uganda consisted of two cultivars susceptible to nematodes, a plantain, Gonja manjaya and an East African Highland banana, Mbwazirume, plus a nematode resistant dessert banana, Yangambi km5. A comparative approach included adding a mixed population of Radopholus similis, Helicotylenchus multicinctus and Meloidogyne spp. to the soil around half the plants of each cultivar prior to field planting. Measurements of LAI were made fortnightly from 106 days post-planting over two successive cropping cycles. The highest mean LAI during the first cycle for Gonja manjaya was suppressed to 74.8±3.5% by the addition of nematodes, while for Mbwazirume the values were reduced to 71.1±1.9%. During the second cycle these values were 69.2±2.2% and 72.2±2.7%, respectively. Reductions in LAI values were validated as due to the biotic stress by assessing nematode numbers in roots and the necrosis they caused at each of two harvests and the relationship is described. Yield losses, including a component due to toppled plants, were 35.3% and 55.3% for Gonja manjaya and 31.4% and 55.8% for Mbwazirume, at first and second harvests respectively. Yangambi km5 showed no decrease in LAI and yield in the presence of nematodes at both harvests. LAI estimated by hemispherical photography provided a rapid basis for detecting biotic growth checks by nematodes on bananas, and demonstrated the potential of the approach for studies of growth checks to other tall crop plants caused by biotic or abiotic stresses.     

Evaluation of an improved RNA/DNA quantification method in a common carp (Cyprinus carpio Linnaeus 1758) larval feeding trial with Artemia,two nematodes (Panagrellus redivivus Linnaeus 1758, Panagrolaimus sp. Fuchs 1930) and dry feed

The RNA/DNA ratio commonly used as proxy for the nutritional condition of fish larvae is affected by RNA degradation during analysis. For evaluation of two strategies to improve RNA integrity, a three‐week feeding trial was carried out to assess the suitability of two nematode species (fam. Panagrolaimidae) as feed for newly hatched carp larvae (Cyprinus carpio) in comparison to Artemia nauplii (Artemia sp.) and a commercial dry feed. Aiming for an increased reproducibility of RNA/DNA determination, a high‐salt inactivation (RNA later) as well as a targeted approach with a recombinant RNase inhibitor were compared to the classical protocol using lab chip technology. Improved RNA integrity was observed with high‐salt inactivation when compared with a strategy applying a specific RNase inhibitor or the classic protocol. Carp larvae fed Artemia for 2 weeks and then dry feed for 1 week revealed the best overall growth performance as well as survival [83.0 ± 35.2 mg fresh weight (FW), 20.0 ± 2.4 mm total length (TL), 86.6 ± 11.7% survival]. Larvae fed the nematode species Panagrellus redivivus for 1 week and subsequently dry feed for 2 weeks (37.4 ± 29.1 mg FW, 14.7 ± 2.8 mm TL, 76.0 ± 6.0% survival) performed better than larvae fed with dry feed alone (28.2 ± 29.6 mg FW, 14.3 ± 2.9 mm TL, 54.3 ± 14.2% survival) or those receiving Panagrellus for 2 weeks. Between both nematode species, Panagrellus was a better feed with regard to growth performance and survival. RNA/DNA ratios ranged between 0.65 ± 0.27 (8 days post‐hatch) and 1.96 ± 0.63 (22 days post‐hatch) and were in the same treatment order as the other growth parameters. RNA/DNA ratios were significantly correlated with the growth rate, and decreasing RNA/DNA ratios in larger larvae may reflect decreasing growth rates with size rather than decreased nutritional status. Here, an improved RNA/DNA ratio protocol is presented in a feeding trial that reveals the suitability of nematodes as a first feed for common carp larvae.     

Evaluation of legume intercrops on the population dynamics and damage level of burrowing nematode (Radopholus similis) in banana (Musa spp.)

Radopholus similis is the most destructive plant parasitic nematode in banana production systems. A glasshouse experiment was carried out to evaluate the effect of legume intercrops on R. similis population and damage level in banana. A trial was laid out in a randomised complete block design with five treatments and five replications. The treatments were banana/cowpea (Vigna unguiculata) intercrop, banana/sunn hemp (Crotalaria juncea) intercrop, sole banana, sole banana with nematicide (Fenamiphos) and sole cowpea. Sunn hemp intercrop was suppressing R. similis population densities and reduced banana root damage the most compared to tested treatments. The legume intercropped banana plants had a significantly higher fresh root mass as the sole-cropped banana with nematicide while the sole-cropped banana without nematicide had significantly lowest fresh root mass. Sunn hemp and cowpea legumes are recommended for adoption by smallholder banana farmers as alternatives to nematicide use.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

Mechanisms of resistance in the rice cultivar Manikpukha to the rice stem nematode Ditylenchus angustus

The incompatible interaction between the rice cultivar Manikpukha and the rice stem nematode Ditylenchus angustus has been reported recently. This research focuses on the underlying mechanisms of resistance in Manikpukha. Invasion, post‐infection development and reproduction of D. angustus were compared in compatible and incompatible interactions to identify the stage in which resistance occurs. The results indicate that resistance in Manikpukha is associated with reduced development and reproduction, implying that resistance acts post‐invasion. We studied the possible involvement of three classical defence hormones, salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), in response to infection in a compatible interaction using biosynthesis/signalling‐deficient transgenic rice lines. All three hormones appear to have an influence on the basal defence of Nipponbare against the stem nematode. Although hormone application increases basal defences, expression studies and hormone analyses after nematode infection in Manikpukha did not show a clear involvement of the hormone defense pathways for SA, ET and JA. However, it seems that OsPAL1 plays a pivotal role in resistance, indicating that the phenylpropanoid pathway and its products might be key players in the incompatible interaction. Lignin measurement showed that, although basal levels are similar, Manikpukha had a significantly higher lignin content on nematode infection, whereas it was decreased in the susceptible cultivar. The results presented here show that SA, ET and JA are involved in basal defences, but the resistance of Manikpukha against D. angustus probably relies on products of the phenylpropanoid pathway.     

An endosymbiotic bacterium in a plant-parasitic nematode: Member of a new Wolbachia supergroup

Wolbachia is an endosymbiotic bacterium widely present in arthropods and animal-parasitic nematodes. Despite previous efforts, it has never been identified in plant-parasitic nematodes. Random sequencing of genes expressed by the burrowing nematode Radopholus similis resulted in several sequences with similarity to Wolbachia genes. The presence of a Wolbachia-like endosymbiont in this plant-parasitic nematode was investigated using both morphological and molecular approaches. Transmission electron microscopy, fluorescent immunolocalisation and staining with DAPI confirmed the presence of the endosymbiont within the reproductive tract of female adults.16S rDNA, ftsZ and groEL gene sequences showed that the endosymbiont of R. similis is distantly related to the known Wolbachia supergroups. Finally, based on our initial success in finding sequences of this endosymbiont by screening an expressed sequence tag (EST) dataset, all nematode ESTs were mined for Wolbachia-like sequences. Although the retained sequences belonged to six different nematode species, R. similis was the only plant-parasitic nematode with traces of Wolbachia. Based on our phylogenetic study and the current literature we designate the endosymbiont of R. similis to a new supergroup (supergroup I) rather than considering it as a new species. Although its role remains unknown, the endosymbiont was found in all individuals tested, pointing towards an essential function of the bacteria.     

Effects of Glomus intraradices on the reproduction of the burrowing nematode (Radopholus similis) in dixenic culture

The effects of Glomus intraradices on the reproduction of the burrowing nematode Radopholus similis were studied under dixenic culture conditions. The life cycles of both the arbuscular mycorrhizal fungus (AMF) and the nematode were completed in presence of each other and a transformed carrot root as host. The AMF suppressed the R. similis population by almost 50% and thus increased protection of the root against the nematode. This reduction was significant for both females and males within roots. There was no correlation between nematode population density and either AMF internal root colonization, external hyphal development or spore production. These results demonstrate that the dixenic system, although artificial, is a valuable tool for studying AMF–nematode interactions, complementing the classical experimental approaches. Accepted: 6 February 2001     

Transgenic potatoes for potato cyst nematode control can replace pesticide use without impact on soil quality

Current and future global crop yields depend upon soil quality to which soil organisms make an important contribution. The European Union seeks to protect European soils and their biodiversity for instance by amending its Directive on pesticide usage. This poses a challenge for control of Globodera pallida (a potato cyst nematode) for which both natural resistance and rotational control are inadequate. One approach of high potential is transgenically based resistance. This work demonstrates the potential in the field of a new transgenic trait for control of G. pallida that suppresses root invasion. It also investigates its impact and that of a second transgenic trait on the non-target soil nematode community. We establish that a peptide that disrupts chemoreception of nematodes without a lethal effect provides resistance to G. pallida in both a containment and a field trial when precisely targeted under control of a root tip-specific promoter. In addition we combine DNA barcoding and quantitative PCR to recognise nematode genera from soil samples without microscope-based observation and use the method for nematode faunal analysis. This approach establishes that the peptide and a cysteine proteinase inhibitor that offer distinct bases for transgenic plant resistance to G. pallida do so without impact on the non-target nematode soil community.     

Pseudomonas fluorescens mediated suppression of Meloidogyne incognita infection of cowpea and tomato

Plant growth-promoting rhizobacterium, Pseudomonas fluorescens strain BICC602 suppresses root-knot nematode (Meloidogyne incognita) by enhancing defence mechanism leading to induced systemic resistance in cowpea (Vigna unguiculata) cv. L.Walp. and tomato (Solanum lycopersicum) cv. Pusa Ruby. In cowpea, the soil treatment proved more effective than foliar spray on root galling and eggs in roots. However, which factors are necessary in the induction of resistance response in plants against nematodes by BICC602 is not yet known. Salicylic acid (SA) production by some bacteria acts as endogenous signal for the activation of certain plant defence responses. In a split-root trial with tomato as a host plant and M. incognita as challenging parasite, BICC602 induces systemic resistance in tomato plants. Based on the results, it is assumed that P. fluorescens-induced resistance against M. incognita in cowpea and tomato is made either through SA-dependent or SA-independent transduction pathway.     

阿维菌素对南方小花蝽抑制烟蚜的影响

在室内分别测定了阿维菌素类杀虫剂1% 9512-1乳油和虫螨克对南方小花蝽Orius similis的毒力,研究了无阿维菌素介入后南方小花蝽对烟蚜Myzus persicae捕食效应所受的影响,结果表明：阿维菌素类杀虫剂对南方小花蝽具有一定毒力,不同虫态对阿维菌素的敏感性有差异,卵的敏感性明显低于若虫和成虫;不同的阿维菌素类药剂对南方小花蝽的毒力存在差异,9512-1对南方小花蝽卵、若虫及成虫的LC_５０依次为11.2743±3.8613、2.6584±1.2139、2.4508±1.3266 mg·L^-１,虫螨克则为10.8699±3.7653、6.4422±2.8596、6.4738±3.0124 mg·L^-１;阿维菌素显著降低了南方小花蝽对烟蚜的捕食量和捕食能力,也降低了同种个体间的竞争能力;阿维菌素对南方小花蝽若虫的发育速率有显著的抑制作用,但对发育速率与取食量的线性关系无明显影响,而且对存活个体成虫期的产卵量无显著影响。     

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

The general potential of plant cystatins for the development of insect‐resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of digestive cysteine protease activities. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet assay with cystatin‐infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC‐I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z‐Phe‐Arg‐MCA‐hydrolyzing (cathepsin L–like) and Z‐Arg‐Arg‐MCA‐hydrolyzing (cathepsin B–like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the cysteine protease inhibitor E‐64 and to different plant cystatins including OsCYS1. In line with the broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatin‐infiltrated banana stem disks. These promising results, which illustrate the susceptibility of C. sordidus to plant cystatins, are discussed in the light of recent hypotheses suggesting a key role for cathepsin B–like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera. © 2009 Wiley Periodicals, Inc.