Daniel Coyne

Resistance to the rice nematodes Heterodera sacchari, Meloidogyne graminicola and M. incognita in Oryza glaberrima and O. glaberrima x O. sativa interspecific hybrids

Rice species Oryza glaberrima and O. sativa and interspecific hybrids produced at the West African Rice Development Association in Cote dIvoire, were screened for resistance to Heterodera sacchari, Meloidogyne graminicola and M. incognita R2. H. sacchari screening was done in field and pot experiments in Cote dIvoire. Non-indigenous species were screened in glasshouse tests in the UK. All O. glaberrima genotypes were resistant to H. sacchari from Cote dIvoire and Ghana, M. graminicola from the Philippines and M. incognita R2. In pot tests, the number of females which developed on these genotypes was zero for H. sacchari and < 3 for both species of Meloidogyne. Within lines of O. sativa, variation in female numbers (P 0.05) was observed, but all were susceptible both to H. sacchari and to M. graminicola. However, six cultivars and lines of O. sativa were less susceptible (P 0.05) to M. incognita R2 than the check cultivar IDSA 6. Of 14 progeny from O. sativa cv. WAB56-104 x O.glaberrima line CG14, two WAB450-I-B-P-105 and WAB450-I-B-P-160, were resistant to H.sacchari from Cote dIvoire. These lines and two others were also less susceptible (P 0.01) to M. graminicola than the O. sativa parent. The susceptibility of some of the progeny to both H. sacchari and M. graminicola and M. incognita was the same as that of the O. sativa parent. Of ten further progeny, screened against H. sacchari from Ghana, WAB450-25-1-10 was resistant. The expression of resistance in the interspecific progeny suggests that resistance to H. sacchari is qualitative, whilst that to Meloidogyne graminicola is quantitative. Resistance to M. incognita R2 was found in O. glaberrima, O. sativa and in one hybrid progeny. None of the species or progeny was resistant to Pratylenchus zeae and there were no significant differences in field population densities of P. zeae, Mesocriconema onoensis or Helicotylenchus dihystera. Resistance aux nematodes Heterodera sacchari, Meloidogyne graminicola et M. incognita chez Oryza glaberrima et chez des hybrides interspecifiques O. glaberrima x O. sativa - Les especes de riz Oryza glaberrima et O. sativa ainsi que des hybrides interspecifiques produits en Cote dIvoire par lAssociation pour le Developpement de la Riziculture en Afrique de lOuest ont ete testes pour leur resistance envers les nematodes Heterodera sacchari, Meloidogyne graminicola et M. incognita R2. Les tests relatifs a H. sacchari ont ete realises, tant en champ quen pots, en Cote dIvoire. Ceux concernant les especes non indigenes lont ete en serre, en Grande Bretagne. Tous les genotypes de O. glaberrima sont resistants aux H. sacchari provenant de Cote dIvoire et du Ghana ainsi qua M. graminicola provenant des Philippines et a M. incognita R2. Lors des tests en pot le nombre de femelles produites sur ces genotypes sont de zero pour H. sacchari et de moins de 3 pour les deux especes de Meloidogyne. Concernant les lignees de O. sativa, une variabilite (P 0,05) a ete observee dans le nombre de femelles produites, mais toutes ces lignees sont sensibles a H. sacchari et M. graminicola. Cependant six cultivars ou lignees de O. sativa sont moins sensibles (P 0,05) a M. incognita R2 que le cultivar temoin IDSA 6. Parmi 14 descendances du croisement O. sativa cv. WAB56-104 x O. glaberrima lignee CG14, deux WAB450-I-B-P-105 et WAB450-I-B-P-160, sont resistances a H. sacchari provenant de Cote dIvoire. Ces lignees, ainsi que deux autres, sont moins sensibles (P 0.01) a M. graminicola que les O. sativa parents. La sensibilite de quelques unes de ces descendances a H. sacchari et aux deux especes de Meloidogyne etait identique a celle des parents. Parmi dix autres descendances ulterieures testees envers H. sacchari du Ghana, WAB450-25-1-10 sest montre resistant. Lexpression de la resistance dans la descendance interspecifique suggere que cette resistance est qualitative pour H. sacchari et quantitative pour M. graminicola. Une resistance a M. incognita a ete decelee chez O. sativa et O. glaberrima ainsi que dans la descendance dun de leurs hybrides. Aucune des especes ou des descendances na montre de resistance envers Pratylenchus zeae et il nexiste aucune difference significative dans les densites de populations au champ de P. zeae, Mesocriconema onoensis ou Helicotylenchus dihystera.

Effect of endophytic Beauveria bassiana on populations of the banana weevil, Cosmopolites sordidus, and their damage in tissue-cultured banana plants

The entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) survives as an endophyte in a wide range of plants, offering substantial protection against tunneling insect pests. Although current research indicates that the fungus can live as an endophyte in tissue‐cultured banana plants, there is no information on the efficacy of endophytic B. bassiana against the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae). Our study aimed at determining the effects of endophytic B. bassiana on oviposition rate and populations of the banana weevil, and their plant damage. Screenhouse studies were undertaken in which 8‐week‐old B. bassiana‐inoculated tissue‐cultured banana plants [cultivar Kibuzi, Musa spec. (Musaceae), genome group AAA‐EA] were infested with female banana weevils. Five days after plant infestation with the banana weevil, there was no effect of B. bassiana on oviposition rate and egg hatchability. However, after 15 weeks, the presence of B. bassiana as an endophyte in banana tissues greatly reduced banana weevil populations and their damage to plants. Between 53.4 and 57.7% of the banana weevil adults died because of B. bassiana infection, resulting in a reduction of plant damage by 29.1–62.7% depending on plant part. Beauveria bassiana was re‐isolated from different plant parts, but to a greater extent from the roots and rhizomes than from pseudostem bases. Artificial inoculation of B. bassiana in banana plants caused no negative impact on banana growth. Our study demonstrated for the first time that endophytic B. bassiana reduced populations of C. sordidus and the plant damage they cause, and can thus be an alternative method for effective delivery of this fungus to banana plants for the management of C. sordidus.

In vitro antagonism of endophytic Fusarium oxysporum isolates against the burrowing nematode Radopholus similis

Radopholus similis is one of the key pests of banana worldwide. In this study, nine endophytic Fusarium oxysporum isolates were screened for the production of secondary metabolites antagonistic to R. similis in culture. Undiluted and diluted culture filtrates were tested against motile stages and eggs of R. similis. All isolates tested demonstrated in vitro antagonistic activity, causing paralysis of R. similis motile stages. The percentage of paralysed nematodes increased with increase in the length of exposure time to culture filtrates. After 24 h exposure in culture filtrates up to 100% of the treated nematodes were paralysed compared to 26.5% in the control treatments. Nematode mortality rates after 24 h exposure in culture filtrates ranged from 76.4% to 100%. Paralysis was reversible at lower filtrate concentrations. Radopholus similis males were more sensitive to culture filtrates than females. Culture filtrates of all isolates demonstrated inhibitory effects on hatching of R. similis eggs. The results demonstrate the potential for using endophytic F. oxysporum as biological control agents against R. similis and for toxic derivatives from their secondary metabolism to be used as potential nematicides.

Micro-plot evaluation of the yield reduction potential of Pratylenchus coffeae, Helicotylenchus multicinctus and Meloidogyne javanica on plantain cv. Apantu-pa (Musa spp., AAB-group) in Ghana

The damage potential of the plant-parasitic nematodes Pratylenchus coffeae, Helicotylenchus multicinctus and Meloidogyne javanica, was assessed on plantain (Musa spp., AAB-group) cv. Apantu-pa in microplots in Ghana. Hot water treated suckers, planted in 3 l plastic bags containing sterilised soil, were inoculated 1 month after planting with a single nematode species or a nematode species mixture; controls were not inoculated. The initial single species inocula consisted of 1000 or 10 000 nematodes per plant, whilst the initial species mixture inoculum consisted of 3000 nematodes of each nematode species per plant. Two months after planting, the suckers were transplanted into micro-plots (0.7 m3 concrete containers filled with sterilised soil). Inoculation of single species at either density resulted in lower (P ≤ 0.05) bunch weights of the mother plants (between 23-33% lower) than the noninoculated control plants, whilst bunch weights of plants inoculated with the species mixture were 18% lower (P ≤ 0.05). Nematode damage indices (% dead roots, root necrosis and sucker corm lesions) were more severe in P. coffeae inoculated treatments. There was no difference between inoculated treatments and the control in plant growth parameters (days to flowering, number of standing leaves, height, girth, number of suckers) of the mother plant at flowering and harvest. Plant toppling occurred only in, and in all, treatments involving P. coffeae, with up to 60% of bunch-carrying plants toppled in the most affected treatment (inoculation of 10 000 nematodes per plant). Therefore, projected yields per ha were low in P. coffeae inoculated treatments: 41, 73 and 65% lower than the control for inoculation of 1000, 10 000 and 3000 (in species mixture) individuals, respectively. Inoculation with 10 000 H. multicinctus or M. javanica per plant resulted in yield losses of 26 and 30%, respectively. This study suggests that P. coffeae is likely to be the most important biotic constraint to plantain production in Ghana. Nematodes have been viewed as the major biotic constraint to plantain production in the country, and P. coffeae is the most widespread and abundant nematode species on plantain in Ghana. The results further demonstrate that H. multicinctus and M. javanica can cause considerable yield reduction in plantain.

Efficacy of chemical and fluorescent protein markers in studying plant colonization by endophytic non-pathogenic Fusarium oxysporum isolates

In studying plant colonization by inoculated Fusarium oxysporum endophytes, it is important to be able to distinguish inoculated isolates from saprophytic strains. In the current study, F. oxysporum isolates were transformed with the green (GFP) and red fluorescent protein (DsRed) genes, and benomyl- and chlorate-resistant mutant isolates were also developed. The benomyl- and chlorate-resistant mutants, and the fluorescently labelled transformants, were able to grow on potato dextrose agar amended with 20xa0mg Benlate®xa0l−1, 30xa0g chloratexa0l−1 and 150xa0μg hygromycinxa0ml−1, respectively. Single spores of all mutants remained stable after several transfers on non-selective media. Most mutants and transformants produced colony diameters that did not differ significantly from that of their wild-type progenitors after 7xa0days of growth on non-selective media. Few mutants, however, had growth rates that were either slower or faster than for their wild-types. Plant colonization studies showed that root and rhizome tissue colonization by most benomyl- and chlorate-resistant mutants was similar to that of their wild-type isolates. Unlike GFP transformants, DsRed transformants were difficult to visualize in planta. Both the mutants and transformants can be used for future studies to investigate colonization, distribution and survival of biocontrol F. oxysporum endophytes in banana plants.

Effect of endophytic Fusarium oxysporum on root penetration and reproduction of Radopholus similis in tissue culture-derived banana ( Musa spp.) plants

In Uganda, banana (Musa spp.) production is constrained by the burrowing nematode Radopholus similis. Non-pathogenic, endophytic Fusarium oxysporum has been isolated from healthy banana plants, and several strains have shown potential as biological control agents against R. similis. Plant infection by R. similis can be characterised in three steps: host searching, root penetration and reproduction. In this study, we investigated the effects of three endophytic F. oxysporum strains (Eny1.31i, Eny7.11o and V5w2) on root penetration and reproduction of R. similis in tissue culture-derived banana plants. The number of R. similis that penetrated roots of endophyte-inoculated and untreated plants was not influenced by the endophytes in either laboratory or screenhouse experiments. However, R. similis reproduction was reduced by endophytes. Strain V5w2 gave the greatest suppression of R. similis reproduction. The results of this study imply that antagonism of endophytic F. oxysporum against R. similis in banana plants is post-infectional and mediated through disruption of nematode reproduction.

Screenhouse and Field Persistence of Nonpathogenic Endophytic Fusarium oxysporum in Musa Tissue Culture Plants

Two major biotic constraints to highland cooking banana (Musa spp., genome group AAA-EA) production in Uganda are the banana weevil Cosmopolites sordidus and the burrowing nematode Radopholus similis. Endophytic Fusarium oxysporum strains inoculated into tissue culture banana plantlets have shown control of the banana weevil and the nematode. We conducted screenhouse and field experiments to investigate persistence in the roots and rhizome of two endophytic Fusarium oxysporum strains, V2w2 and III4w1, inoculated into tissue-culture banana plantlets of highland cooking banana cultivars Kibuzi and Nabusa. Re-isolation of F. oxysporum showed that endophyte colonization decreased faster from the rhizomes than from the roots of inoculated plants, both in the screenhouse and in the field. Whereas rhizome colonization by F. oxysporum decreased in the screenhouse (4–16xa0weeks after inoculation), root colonization did not. However, in the field (17–33xa0weeks after inoculation), a decrease was observed in both rhizome and root colonization. The results show a better persistence in the roots than rhizomes of endophytic F. oxysporum strains V2w2 and III4w1.

Morphological and molecular characterisation and diagnostics of some species of Scutellonema Andrássy, 1958 (Tylenchida: Hoplolaimidae) with a molecular phylogeny of the genus

Scutellonema spp. are widely distributed across tropical and subtropical regions of the world and are associated with numerous agricultural and horticultural crops. Identification of many Scutellonema species is not always reliable, in part because many species share very similar diagnostic characters. In this study, we provide morphological and molecular characterisation of S. brachyurus from the USA and South Africa, S. bradys from Nigeria and three unidentified species from California, USA, New Zealand and Burkina Faso. Morphological descriptions, measurements, light and scanning electron microscopic photos and drawings are given for S. brachyurus. Females of S. brachyurus from the USA (type A) and South Africa (type B) showed a significant variation in the number of sectors and blocks on the lip annuli, ranging from about 4-12 and from 8-20, respectively. Molecular analysis using the D2-D3 of 28S rRNA, ITS rRNA and the COI mtDNA gene sequences revealed two distinct genotypes within S. brachyurus samples: type A (samples from USA, Italy, Korea, Taiwan) and type B (South Africa). Multivariate analyses determined that S. brachyurus from the USA and Taiwan (type A) differed from that from South Africa (type B) mainly in body, tail and DGO lengths, and ratios b′, c′, c and V. Phylogenetic relationships within Scutellonema are given as inferred from the analyses of the D2-D3 of 28S rRNA, ITS rRNA and the COI mtDNA gene sequences. PCR-RFLP diagnostic profiles and PCR with species-specific primers are developed for the studied Scutellonema species.

Differential effect of hot water treatment on whole tubers versus cut setts of yam (Dioscorea spp.)

BACKGROUNDnThe use of thermotherapy or hot water treatment (HWT) is recommended for the management of plant-parasitic nematodes and other pathogens for a range of planting material, especially vegetatively propagated crops including yams, Dioscorea spp. The sprouting (germination) and consequent viability of yam following HWT, however, appear to be influenced by the post-treatment method of planting (whole or cut setts) and cultivar. The present study was established to evaluate the sensitivity of the most popular yam cultivars in Benin and Nigeria, West Africa, to HWT at 50-53 degrees C for 20 min.nnnRESULTSnSprouting of both setts and whole tubers of most cultivars was affected by HWT. Across experiments, 47% of HWT material, compared with 61% of non-HWT material, sprouted over 8 weeks. When cut into setts, 41% of HWT or untreated tubers sprouted, compared with 72% of whole tubers. Whole, untreated tubers had highest sprouting rates (84%), and setts following HWT had the lowest (38%). Yam planting material was also not completely free of parasitic nematodes following HWT. The reaction to HWT or cutting was highly cultivar specific.nnnCONCLUSIONnYam cultivars vary in their sensitivity to hot water therapy. Care is therefore advised in selecting yam cultivars for HWT, especially when using cut setts.

Monoxenic culture of Pratylenchus sudanensis on carrot disks, with evidence of differences in reproductive rates between geographical isolates

Sterile carrot disks have been used successfully for monoxenic culture of root lesion nematodes such as Pratylenchus brachyurus (Moody et al., 1973) and P. vulnus (Townson & Lear, 1982), and excised maize roots have been used to rear P. penetrans (Tiner, 1960) and P. zeae (Jordaan & De Waele, 1988). Recent reports have indicated that P. sudanensis is causing damage to yams (Dioscorea spp.) in Uganda (Mudiope, 1999; Coyne et al., 2003). Information on the biology and epidemiology of P. sudanensis is very limited. It has been associated with over 20 plant species in Sudan, however, where it is viewed as an economically damaging pest of cotton (Saadabi, 1985). Other favourable hosts included sorghum, pigeon pea and lubia bean. Wheat and groundnut were viewed as non-hosts. In order to investigate further the biology of this nematode, a readily available source of nematode cultures would be needed. Therefore, sterile carrot disk and excised maize root methods were assessed for their ability to support mass culture of P. sudanensis.