Napier Grass Stunt Disease Research Articles

Napier grass stunt disease: Effector gene prediction

Napier grass stunt disease: Effector gene prediction

Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae

Napier grass Stunt Disease (NSD) is a severe disease of Napier grass (Pennisetum purpureum) in Eastern Africa, caused by the leafhopper-transmitted bacterium Candidatus Phytoplasma oryzae. The pathogen severely impairs the growth of Napier grass, the major fodder for dairy cattle in Eastern Africa. NSD is associated with biomass losses of up to 70% of infected plants.Diagnosis of NSD is done by nested PCR targeting the phytoplasma DNA, which is difficult to perform in developing countries with little infrastructure. We report the development of an easy to use, rapid, sensitive and specific molecular assay for field diagnosis of NSD. The procedure is based on recombinase polymerase amplification and targets the imp gene encoding a pathogen-specific immunodominant membrane protein. Therefore we followed a two-step process. First we developed an isothermal DNA amplification method for real time fluorescence application and then transferred this assay to a lateral flow format. The limit of detection for both procedures was estimated to be 10 organisms. We simplified the template preparation procedure by using freshly squeezed phloem sap from Napier grass. Additionally, we developed a laboratory serological assay with the potential to be converted to a lateral flow assay. Two murine monoclonal antibodies with high affinity and specificity to the immunodominant membrane protein IMP of Candidatus Phytoplasma oryzae were generated. Both antibodies specifically reacted with the denatured or native 17 kDa IMP protein. In dot blot experiments of extracts from infected plant, phytoplasmas were detected in as little as 12,5 μg of fresh plant material.

Screening Napier grass accessions for resistance to Napier grass stunt disease using the loop-mediated isothermal amplification of DNA (LAMP)

Napier grass stunt (NGS) disease is a major threat to Napier grass cultivation and the smallholder dairy industry in East and Central Africa. The disease is caused by a phytoplasma, which is transmitted by the leafhopper Maiestas banda (Kramer) (Hemiptera: Cicadellidae). The current study was conducted to identify among 65 Napier grass accessions that could be resistant to NGS disease using the loop-mediated isothermal amplification of DNA (LAMP). The accessions were caged with NGS phytoplasma-infected Napier grass as inoculum source and M. banda as the vector. All Napier grass accessions were subjected to phytoplasma testing thereafter 18 phytoplasma negative and five asymptomatic accessions were selected and used in further screening by subjecting the extracted DNA to LAMP. Plant response to the NGS phytoplasma by symptom expression, impact on yield-related parameters and phytoplasma infection was used to evaluate tolerance or resistance over a period of three months. Most Napier grass accessions were susceptible to the disease except plants belonging to accession 16789 which were negative by LAMP. Napier grass accession 16807 was found to be tolerant with 60% plants positive by LAMP and 90% plants symptomless. Accessions 16822 and 16817 had moderate tolerance with one and two plants positive by LAMP, respectively. Accession 16812 was slightly tolerant with 58.3% plants positive by LAMP and 33.3% of the plants showing symptom remission in the second re-growth. This study indicates there could be resistance and tolerance to NGS disease which could be exploited in the development of an integrated management strategy for this disease.

Draft Genome Sequence of "Candidatus Phytoplasma oryzae" Strain Mbita1, the Causative Agent of Napier Grass Stunt Disease in Kenya.

Phytoplasmas are bacterial plant pathogens with devastating impact on agricultural production worldwide. In eastern Africa, Napier grass stunt disease causes serious economic losses in the smallholder dairy industry. This draft genome sequence of “Candidatus Phytoplasma oryzae” strain Mbita1 provides insight into its genomic organization and the molecular basis of pathogenicity.

Napier grass stunt disease prevalence, incidence, severity and genetic variability of the associated phytoplasma in Uganda

The prevalence, incidence and severity of Napier grass stunt disease (NGSD) caused by phytoplasma on Pennisetum purpureum, the main fodder for livestock under intensive and semi-intensive management systems in Uganda were determined following a field survey carried out in 17 districts. A total of 298 Napier grass fields were visited and NGSD status visually assessed and 1192 samples collected for identification and confirmation of the phytoplasma by polymerase chain reaction (PCR) assays using universal primers P1/P6 nested with R16F2n/R16R2n and, tuf primers 890/340 nested with 835 and 400. From these, 221 PCR products were sequenced and sequences aligned. Napier grass stunt disease is widely spread at an epidemic proportion, with the districts at different risk levels. The most affected districts are in central, East and North parts of the country while those in the west are least affected. Sequence alignments and Blast searches showed that the phytoplasma causing NGSD in Uganda belonged to the phytoplasma group 16SrXI, with single nucleotide sequence variants in a few districts. Therefore, there is a need for development of an area wide NGSD management strategy to contain the disease.

Napier grass stunt disease in East Africa: Farmers' perspectives on disease management

Napier or Elephant grass (Pennisetum purpureum), the most important livestock fodder crop in East Africa, is under threat from Napier grass stunt (NGS) disease. This disease is caused by a phytoplasma, which is transmitted by the leafhopper, Maiestas banda (Hemiptera: Cicadellidae). After inoculation, the disease rapidly infects the whole plant causing extensive damage to Napier grass plants. There is therefore a need to develop an integrated management approach for the disease. A survey was conducted in three East African countries (Kenya, Uganda and Tanzania) during which 198 farmers were interviewed using semi-structured questionnaires. The questionnaire addressed the prevalence of NGS, source of planting material and Napier grass cultivars grown. Farmers were also questioned on how they perceived the severity of the disease, management options and on the knowledge of the existence of wild grass hosts of the disease and its vector. Disease prevalence in survey areas was expressed as a percentage of the total 198 fields assessed. Chi-square (χ2), F tests and one-way analysis of variance (ANOVA) were conducted to asses any differences between districts, gender and education levels with regards to the knowledge and perceptions of NGS in the three countries. The prevalence of NGS ranged from 33% in Uganda to 95.7% in Kenya with an average of 55.1%. Among the farmers interviewed, 49.5% were able to recognize NGS symptoms. Most farmers did not have effective management approaches for the disease but cited a variety of measures including roguing and introduction of alternative fodder grasses that could potentially form part of an integrated management approach for the disease. Sedge grass (Cyperus sp.) and Star grass (Cynodon dactylon) were listed by the respondents as the likely hosts of stunt diseases caused by phytoplasma. It has been long suspected that weeds could play a role in the spread of phytoplasma by acting both as reservoirs from which healthy plants could be re-infected and also as hosts for the vectors. Since there is no well-established control method for NGS, the majority of farmers uproot infected plants and replant with new ones to lower the infection pressure by providing fewer infected plants for vectors to feed on. This pinpoints the need for farmers' awareness and the need to develop resistant Napier grass cultivars as a management option.

Temporal Dynamics of Napier Grass Stunt Disease as Influenced by Napier Grass Clones and Initial Inoculum

Napier grass stunt disease (NGSD) is the main biotic factor limiting Napier grass production in the East African reg ion. Its management is, however, hampered by inadequate epidemiological information. This study determined the temporal spread of NGSD in Napier grass fields. A field experiment was setup at National Crops Resources Research Institute, Namulonge in Uganda to determine the influence of initial inoculum and clones on the spread of NSD in the field. The experiment was arranged in a randomized Complete Block Design and replicated 4 times. The initial inoculum levels used were 0%, 10%, 20% and 30% while the clon es included KW4, local/wild type and P99, respectively. Napier grass stunt disease incidence data was recorded at

Identification of Potential Vectors and Alternative Plant Hosts for the Phytoplasma Associated with Napier Grass Stunt Disease in Ethiopia

AbstractNapier grass (Pennisetum purpureum), the most important forage crop in East Africa, has recently been affected by a devastating disease named Napier Grass Stunt (NGS). A phytoplasma of group 16SrI has been associated with NGS in Kenya and Uganda, whereas in Ethiopia, group 16SrIII was previously identified in NGS affected fields. However, no insect vectors or alternative hosts have been recorded for NGS in East Africa. During 2005, surveys were conducted at NGS‐affected plantations of Debre‐Zeit and Zwai field stations in Addis Ababa. Leaf samples were collected from weeds located in and surrounding the NGS‐affected areas. Leafhopper species were also surveyed by vacuum sampling in a search for natural phytoplasma vectors. Total DNA was extracted from plants and insects, and used as a template in nested polymerase chain reaction (nPCR) with universal 16S rRNA phytoplasma primers. Restriction fragment length polymorphism (RFLP), sequencing of PCR products and phylogenetic analysis were conducted for a finer identification and characterization of the phytoplasma associated with NGS. A 16SrIII‐A phytoplasma with 100% of identity in the 16S rRNA sequence with that of the previously identified one in Napier Grass (accession no. DQ305977) was identified from alfalfa, Medicago sativa (accession no. DQ305982), Cynodon dactylon (accession no. DQ3058983), Exitianus sp. (DQ305980) and Leptodelphax dymas collected in Debre Zeit (accession no. DQ305979) and Zwai (accession no. DQ305978). These findings suggest that M. sativa and Cy. dactylon are alternative reservoirs, and Exitianus sp. and L. dymas, potential vectors of the 16SrIII‐A phytoplasma, which may have epidemiological implications in spreading NGS in Ethiopia.

First report of a 16SrXI group phytoplasma (Candidatus Phytoplasma oryzae) associated with Napier grass stunt disease in Uganda

Napier grass (Pennisetum purpureum), also known as elephant grass, is an important fodder crop in Uganda. The forage has been increasingly used in intensive (zero grazing) and semi-intensive dairy cattle production systems, in order to meet the increasing demand for milk in Uganda. Napier grass stunt disease, causing short internodes, bushy appearance, yellow to purple streaking and a low biomass yield, has spread among smallholder dairy farmers in Uganda since it was first observed in 2001 (Alacai et al., 2004). The disease has also been reported from western Kenya and shown to be associated with a phytoplasma (Jones et al., 2004) and in Tanzania (B. Pallangyo, personal communication). Leaf samples of stunted Napier grass were collected in the three major Napier grass-growing areas of Uganda. Air-dried leaf samples were ground in liquid nitrogen and total DNA was extracted using a DNeasy kit according to manufacturer's instructions (Qiagen). Leaves without symptoms were used as negative control. The presence of phytoplasma in plants with symptoms was confirmed by a real-time PCR method according to Christensen et al. (2004). Seven of these positive samples were sequenced in the 16S rRNA gene (deposited in GenBank Acc. No. EF012650) and the 16S/23S intergenic spacer (Acc. No. EF012649). No sequence variation was found. The sequences were 99·3% identical to the 16S rRNA sequences from the phytoplasma associated with Napier grass stunt from Kenya, which belongs to the 16SrXI rice yellow dwarf group (Candidatus Phytoplasma oryzae; Jones et al., 2004). This is the first report of a 16SrXI Group phytoplasma associated with Napier grass stunt disease in Uganda.