Danny Coyne

Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution.

The polyphagous parthenogenetic root-knot nematodes of the genus Meloidogyne are considered to be the most significant nematode pest in sub-tropical and tropical agriculture. Despite the crucial need for correct diagnosis, identification of these pathogens remains problematic. The traditionally used diagnostic strategies, including morphometrics, host-range tests, biochemical and molecular techniques, now appear to be unreliable due to the recently-suggested hybrid origin of root-knot nematodes. In order to determine a suitable barcode region for these pathogens nine quickly-evolving mitochondrial coding genes were screened. Resulting haplotype networks revealed closely related lineages indicating a recent speciation, an anthropogenic-aided distribution through agricultural practices, and evidence for reticulate evolution within M. arenaria. Nonetheless, nucleotide polymorphisms harbor enough variation to distinguish these closely-related lineages. Furthermore, completeness of lineage sorting was verified by screening 80 populations from widespread geographical origins and variable hosts. Importantly, our results indicate that mitochondrial haplotypes are strongly linked and consistent with traditional esterase isozyme patterns, suggesting that different parthenogenetic lineages can be reliably identified using mitochondrial haplotypes. The study indicates that the barcode region Nad5 can reliably identify the major lineages of tropical root-knot nematodes.

Dual inoculation of Fusarium oxysporum endophytes in banana: effect on plant colonization, growth and control of the root burrowing nematode and the banana weevil

Abstract The burrowing nematode (Radopholus similis (Cobb) Thorne) and the banana weevil (Cosmopolites sordidus Germar, Coleoptera: Curculionidae) are major pests of banana (Musa spp.) in the Lake Victoria basin region of Uganda. Among biological options to control the two pests is the use of non-pathogenic Fusarium oxysporum Schltdl.: Fries endophytes of banana. We investigated the ability of endophytic F. oxysporum isolates Emb2.4o and V5w2 to control the banana weevil and the burrowing nematode, alone and in combination. Plant colonization by the endophytes was determined by inoculating their chemical-resistant mutants separately and in combination, onto banana roots. Plant growth promotion was determined by measuring plant height, girth, number of live roots and fresh root weight at harvest, and control of the nematode and weevil was determined by challenging endophyte-inoculated plants with the pests 8 weeks after endophyte inoculation. Endophytic root colonization was highest in plants inoculated with both endophytes, compared with those inoculated with only one of the endophytes. Root colonization was better for isolate V5w2 than Emb2.4o. Dually inoculated plants showed a significant increase in height, girth, fresh root weight and number of functional roots following nematode challenge. Nematode numbers in roots were reduced 12 weeks after challenge of 8-week-old endophyte-inoculated plants. Significant reductions in weevil damage were observed in the rhizome periphery, inner and outer rhizomes, compared with endophyte non-inoculated controls. We conclude that dual inoculation of bananas with endophytic isolates Emb2.4o and V5w2 increases root colonization by the endophytes, reduces nematode numbers and weevil damage, and enhances plant growth in the presence of nematode infestation.

Field resistance of transgenic plantain to nematodes has potential for future African food security

Plant parasitic nematodes impose losses of up to 70% on plantains and cooking bananas in Africa. Application of nematicides is inappropriate and resistant cultivars are unavailable. Where grown, demand for plantain is more than for other staple crops. Confined field testing demonstrated that transgenic expression of a biosafe, anti-feedant cysteine proteinase inhibitor and an anti-root invasion, non-lethal synthetic peptide confers resistance to plantain against the key nematode pests Radopholus similis and Helicotylenchus multicinctus. The best peptide transgenic line showed improved agronomic performance relative to non-transgenic controls and provided about 99% nematode resistance at harvest of the mother crop. Its yield was about 186% in comparison with the nematode challenged control non-transgenic plants based on larger bunches and diminished plant toppling in storms, due to less root damage. This is strong evidence for utilizing this resistance to support the future food security of 70 million, mainly poor Africans that depend upon plantain as a staple food.

Effects of nematode infection and mulching on the yield of plantain ( Musa spp., AAB-group) ratoon crops and plantation longevity in southeastern Nigeria

The effects of nematode infection and mulching on plantain cv. Agbagba ( Musa spp., AAB-group, false horn) yield and plantation longevity were examined in a field experiment at the High Rainfall Station of the International Institute of Tropical Agriculture (IITA) at Onne in southeastern Nigeria. The 2nd, 3rd and 4th crop cycles (1st, 2nd and 3rd ratoon, respectively) following the plant crop cycle (mother crop) were examined in nematode inoculated or non-inoculated and mulched or non-mulched treatments. Nematodes ( Radopholus similis , Helicotylenchus multicinctus and, to a lesser extent, Helicotylenchus dihystera , Hoplolaimus pararobustus and Meloidogyne spp.) were inoculated at planting of the mother plant, but were also present in relatively high population densities in the non-inoculated treatments at harvest of the 1st crop cycle. Plants inoculated with nematodes failed to reach harvest and neither did plants in the non-inoculated non-mulched treatments in any ratoon. Only non-inoculated mulched plants reached harvest, producing 0.85, 1.22 and 0.2 Mg ha −1 , respectively in the 1st, 2nd and 3rd ratoon, taking toppled, broken and dead plants into consideration. Mulched plants were larger, had more suckers, survived longer and had healthier root systems compared with non-mulched plants. Damage to roots was greater in the inoculated plants compared with the non-inoculated plants for the 1st and 2nd ratoons but not for the 3rd ratoon. Radopholus similis was most strongly associated with root damage (percentage root necrosis and dead roots), although H. multicinctus population densities were also positively correlated with percentage root necrosis. At flowering of the 1st ratoon, 71% of the inoculated non-mulched plants were dead compared with only 1% of the non-inoculated mulched plants. Helicotylenchus multicinctus remained the most abundant nematode throughout the experiment. Together with R. similis , it comprised over 95% of the plant-parasitic nematode population.

Mitochondrial haplotype-based identification of ethanol-preserved root-knot nematodes from Africa.

The asexual root-knot nematodes (RKNs) (Meloidogyne spp.) exemplified by Meloidogyne incognita are widespread and damaging pests in tropical and subtropical regions worldwide. Comparison of amplification products of two adjacent polymorphic regions of the mitochondrial genome using DNA extracts of characterized RKN strains, including 15 different species, indicate that several species are derived from the same or closely related female lineages. Nevertheless, M. javanica, M. enterolobii, M. incognita, and other key species could each be assigned unique mitochondrial haplotypes based on polymerase chain reaction fragment size and restriction cleavage patterns. M. arenaria isolates did not group as a single haplotype, consistent with other reports of diversity within this species. To test the utility of this assay, we characterized ethanol-preserved samples from 103 single-species isolates from four countries in sub-Saharan Africa (Benin, Nigeria, Kenya, and Tanzania). Mitochondrial haplotypes corresponding to M. javanica and M. incognita were the most prevalent. Samples from western Africa included several instances of M. enterolobii but this species was not detected in samples from East Africa. This protocol provides progress toward a standardized strategy for identification of RKN species from small, preserved samples and a rational starting point for classifying species present in regions where previous knowledge has been limited.

Pratylenchus speijeri n. sp. (Nematoda: Pratylenchidae), a new root-lesion nematode pest of plantain in West Africa.

A new root-lesion nematode, particularly pathogenic to Musa spp. and causing important plantain losses in Ghana, is described and named Pratylenchus speijeri n. sp. The cryptic status of this species within the P. coffeae species complex has been assessed and confirmed in this study. An extensive comparison of the morphological and molecular characteristics of this new species with those of P. coffeae and other related amphimictic species did not result in an unambiguous separation of this species from P. coffeae because only a few morphological features of diagnostic value were found. Sequence and phylogenetic analyses of the D2-D3 expansion segments of the 28S rRNA gene, the ITS rRNA gene and a portion of the hsp90 gene of P. speijeri n. sp. and P. coffeae species complex populations from different sources generated majority consensus BI trees with three major clades: P. speijeri n. sp. from Musa spp. roots in Ghana; unidentified or putative new Pratylenchus sp. C1 from Colocasia esculenta roots in Japan and P. coffeae with non-homogeneous relationships from different hosts and distant geographical areas. These results confirmed the validity of P. speijeri n. sp. as a new taxon and indicated that P. coffeae populations from Colocasia in Japan also need to be considered as a new species. Sequence differences in the ITS were used to design group- and species-specific primers to detect P. speijeri n. sp. and other species of P. coffeae species complex. The use of these species-specific primers for the separation of P. speijeri n. sp., Pratylenchus sp. C1 and P. coffeae has important practical application in breeding programmes for agriculture in West Africa.

Racocetra beninensis from sub-Saharan savannas : a new species in the Glomeromycetes with ornamented spores

A new fungal species is described under the epithet Racocetra beninensis (Racocetraceae, Glomeromycota). It forms white to creamy-white, globose to sub-globose (sometimes oval) glomerospores terminally on sporogenous Cells. Spores measure 195-335 mu m diam and have two spore walls: a three-layered outer wall and a three-layered inner wall. The Outer spore surface is ornamented with rounded wart-like projections that measure 0.9-2.8 x 0.9-3.8 mu m and are spaced (2.2-)4.0-11.0 mu m apart. The germination shield that forms on the outer surface of the inner wall is multiple-lobed (6-10 lobes) and (sub-)hyaline or occasionally yellowing with age. The lobes regularly bear a single germ tube initiation. The fungus differs from other Racocetra species by spore size and color, ornamentation type, and outer spore wall staining reaction. It has been frequently recovered from sites under natural vegetation and newly cultivated or post-harvest yam (Dioscorea spp.) fields in the sub-Saharan Sudan and Guinea savannas of Benin (West Africa).

Measurement of the field response of Musa genotypes to Radopholus Similis and Helicotylenchus Multicinctus and the implications for nematode resistance breeding

Crop growth and damage parameters (plant growth and yield, root damage and nematode population densities), believed to be associated with resistance of Musa genotypes to nematodes under field conditions, were evaluated in a field trial of 24 Musa genotypes inoculated at planting with a combination of Radopholus similis and Helicotylenchus multicinctus with the objective to identify parameters with strong association with nematode resistance and high heritability. Correlation and path analysis of the association between plant growth, yield, root damage and nematode population densities showed a strong negative association between percentage dead roots, percentage root necrosis, R. similis and H. multicinctus population densities and yield. The strongest negative association was observed between percentage dead roots and yield. Broad-sense genotype heritability estimates demonstrated that heritability estimates for percentage dead roots, number of large lesions and nematode population density were most affected by inoculation with nematodes. These results indicate therefore that effective selection for nematode resistance under field conditions could be obtained by using an index, that includes percentage dead roots, the number of large lesions, and nematode population density.

Integrative taxonomy of root-knot nematodes reveals multiple independent origins of mitotic parthenogenesis

During sampling of several Coffea arabica plantations in Tanzania severe root galling, caused by a root-knot nematode was observed. From pure cultures, morphology and morphometrics of juveniles and females matched perfectly with Meloidogyne africana, whereas morphology of the males matched identically with those of Meloidogyne decalineata. Based on their Cox1 sequence, however, the recovered juveniles, females and males were confirmed to belong to the same species, creating a taxonomic conundrum. Adding further to this puzzle, re-examination of M. oteifae type material showed insufficient morphological evidence to maintain its status as a separate species. Consequently, M. decalineata and M. oteifae are synonymized with M. africana, which is herewith redescribed based on results of light and scanning electron microscopy, ribosomal and mitochondrial DNA sequences, isozyme electrophoresis, along with bionomic and cytogenetic features. Multi-gene phylogenetic analysis placed M. africana outside of the three major clades, together with M. coffeicola, M. ichinohei and M. camelliae. This phylogenetic position was confirmed by several morphological features, including cellular structure of the spermatheca, egg mass position, perineal pattern and head shape. Moreover, M. africana was found to be a polyphagous species, demonstrating that “early-branching” Meloidogyne spp. are not as oligophagous as had previously been assumed. Cytogenetic information indicates M. africana (2n = 21) and M. ardenensis (2n = 51–54) to be a triploid mitotic parthenogenetic species, revealing at least four independent origins of mitotic parthenogenesis within the genus Meloidogyne. Furthermore, M. mali (n = 12) was found to reproduce by amphimixis, indicating that amphimictic species with a limited number of chromosomes are widespread in the genus, potentially reflecting the ancestral state of the genus. The wide variation in chromosome numbers and associated changes in reproduction modes indicate that cytogenetic evolution played a crucial role in the speciation of root-knot nematodes and plant-parasitic nematodes in general.

Plant parasitic nematode populations on upland and hydromorphic rice in Côte d’Ivoire: relationship with moisture availability and crop development on a valley slope

Abstract The influence of underlying hydrology and overhead irrigation on plant parasitic nematode populations in upland and hydromorphic rice was examined during three successive seasons on a valley slope at the West Africa Rice Development Association station, Cote d’Ivoire. Nematode population densities and species composition were clearly affected by hydrology. Nematode populations in hydromorphic rice were characterised by Basiria spp., Coslenchus spp. and Filenchus spp. (combined) and Paratrichodorus minor . Upland rice was characterised by Scutellonema clathricaudatum, Pratylenchus zeae, and Mesocriconema tescorum . Rotylenchulus spp. and Heterodera sacchari being concentrated in the lower upland rice area whereas Helicotylenchus spp. and Meloidogyne incognita, were distributed over the hill slope. For many species peak population density shifted, relative to the slope, between sowing and harvest. Excepting the disappearance of rarer species, only H. sacchari population densities were correlated with time, increasing with duration of the experiment. There was little evidence that overhead irrigation had any influence on nematode species composition, but it influenced population densities of ectoparasitic nematodes and had a strong positive influence on yield. Yield was also positively correlated with proximity to the water table and declined as the experiment progressed. Because of the complexity of nematode communities it was difficult to determine the effects of individual species on plant growth characters or yield.