Napier Grass Stunt Research Articles

The Physiological and Biochemical Effects on Napier Grass Plants Following Napier Grass Stunt Phytoplasma Infection

Napier grass stunt (NGS) phytoplasma, a phloem-limited bacterium, infects Napier grass leading to severe yield losses in East Africa. The infected plants are strongly inhibited in growth and biomass production. In this study, phytoplasma-induced morphological changes of the vascular system and physiological changes were analyzed and compared with uninfected plants. The study showed that the phytoplasmas are more abundant in source leaves and range from 103 bacteria/μg total DNA in infected roots to 106 in mature Napier grass leaves. Using microscopical, biochemical, and physiological tools, we demonstrated that the ultrastructure of the phloem and sieve elements is severely altered in the infected plants, which results in the reduction of both the mass flow and the translocation of photoassimilates in the infected leaves. The reduced transport rate inhibits the photochemistry of photosystem II in the infected plants, which is accompanied by loss of chloroplastic pigments in response to the phytoplasma infection stress eventually resulting in yellowing of diseased plants. The phytoplasma infection stress also causes imbalances in the levels of defense-related antioxidants, glutathione, ascorbic acid, reactive oxygen species (ROS), and-in particular-hydrogen peroxide. This study shows that the infection of NGS phytoplasma in the phloem of Napier grass has an impact on the primary metabolism and activates a ROS-dependent defense response.

Napier grass stunt disease: Effector gene prediction

Napier grass stunt disease: Effector gene prediction

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.

Refinement of the Taxonomic Structure of 16SrXI and 16SrXIV Phytoplasmas of Gramineous Plants using Multilocus Sequence Typing.

Phytoplasmas that infect gramineous plants, including Napier grass stunt, sugarcane whiteleaf, sugarcane grassy shoot, and Bermuda grass whiteleaf, have been classified into two closely related groups, 16SrXI and 16SrXIV, based on the 16S ribosomal RNA (rRNA) gene. Subsequently, phytoplasmas associated with coconut and Areca palm in southern India and Sri Lanka have been added into the 16SrXI group. However, the 16S rRNA gene gives relatively poor resolution between these phytoplasmas. In this study, a new set of universal phytoplasma primers that amplify approximately 1 kb of the leucyl transfer RNA synthetase (leuS) gene have been validated on a broad range of phytoplasma taxonomic groups. These have been used along with partial sequences of the secA gene to clarify the taxonomic classification of 16SrXI and 16SrXIV phytoplasmas. Based on this data, the sugarcane whiteleaf and grassy shoot phytoplasmas appear to be the same phytoplasma. The Napier grass stunt phytoplasma forms a distinct group from the Bermuda grass whiteleaf and sugarcane phytoplasmas, suggesting that Napier grass stunt should be in its own 'Candidatus Phytoplasma sp.'. The phytoplasmas associated with coconut and arecanut in southern India and Sri Lanka, which are in the same 16SrXI group, appear in different groups based on secA analysis.

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.

The Significance of Napier Grass Stunt Phytoplasma and Its Transmission to Cereals and Sugarcane

AbstractProduction of Napier grass, Pennisetum purpureum, the most important forage crop in Africa, is severely constrained by Napier grass stunt (NGS) disease. Wild grasses have been identified as alternative hosts for NGS, but there is no record regarding the ability of this phytoplasma to infect cultivated food crops. To assess this threat, six crop species were placed around phytoplasma‐infected Napier grass with the vector Maiestas banda (Hemiptera: Cicadellidae) in transmission cages for a period of 30 days. The crop plants were observed for 3 months after removal from the cages for disease development. Polymerase chain reaction based on the conserved 16S gene, primed by P1/P6‐NapF/NapR nested primer sets, was used to diagnose phytoplasma in test plants, before and after transmission experiments. Pennisetum purpureum showed the highest infection level (81.3%), followed by Saccharum officinarum (56.3%), Eleusine coracana (50%), Sorghum bicolor (43.8%), Oryza sativa (31.3%) and Zea mays (18.8%). All the test plants remained symptomless except sugarcane that exhibited mild to moderate symptoms comprising yellow leaves and bright white or yellow midribs. Napier grass, however, was stunted with small yellow leaves. The study showed that food crops could be infected, suggesting their possible role as sources of inoculum for NGS and their contribution in the spread of the disease in regions where these crops are cultivated. This implies an additional disease cycle could occur for NGS disease, highlighting the challenges to development and implementation of management strategies for the disease.

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

Saving livestock fodder in East Africa: development of a rapid penside diagnostic assay for detection of napier grass stunt phytoplasma

Napier grass stunt is a disease associated with phytoplasmas belonging to the 16SrXI subgroup. The disease affects Pennisetum purpureum, a perennial grass species with significant importance in dairy livestock nutrition and ecological management of pests and parasitic weeds. The present study explores the development, testing and validation of field-friendly diagnostic tools for napier grass stunt phytoplasma detection in East Africa, based on an isothermal assay targeting the gene encoding the immunodominant protein of napier grass stunt phytoplasma.

Phylogenetic analysis identifies a ‘Candidatus Phytoplasma oryzae ’-related strain associated with yellow leaf disease of areca palm (Areca catechu L.) in India

Yellow leaf disease (YLD) with phytoplasmal aetiology is a serious disease of arecanut palm in India. The present study was undertaken to characterize the 16S rRNA and secA gene sequences of the Indian arecanut YLD phytoplasma for 'Candidatus Phytoplasma' species assignment and 16Sr group/subgroup classification. Phytoplasma 16S rRNA genes were amplified using three sets of semi-nested/nested primers, 1F7/7R3-1F7/7R2, 4Fwd/3Rev-4Fwd/5Rev and P1/P7-R16F2n/R16R2, producing amplicons of 491, 1150 and 1250 bp, respectively, from diseased samples. The amplicons were cloned and sequenced. A blast search showed that the sequences had 99 % similarity with sugar cane white leaf phytoplasma (16SrXI) and Napier grass stunt phytoplasma (16SrXI). Phylogenetic analysis based on the 16S rRNA gene revealed the clustering of YLD phytoplasma with the rice yellow dwarf and Bermuda grass white leaf groups. The YLD phytoplasma F2nR2 sequence shared 97.5 % identity with that of 'Candidatus Phytoplasma oryzae' and 97.8 % identity with that of 'Candidatus Phytoplasma cynodontis'. Hence, for finer differentiation, we examined the secA gene-based phylogeny, where the YLD phytoplasma clustered with Napier grass stunt and sugar cane grassy shoot phytoplasmas, both belonging to the rice yellow dwarf group. Hence, we are assigning the Indian arecanut YLD phytoplasma as a 'Candidatus Phytoplasma oryzae'-related strain. Virtual RFLP analysis of a 1.2 kb fragment of the 16S rRNA gene (F2nR2 region) identified the Indian arecanut YLD phytoplasma as a member of 16SrXI-B subgroup. We name the phytoplasma Indian yellow leaf disease phytoplasma, to differentiate it from the Hainan YLD phytoplasma, which belongs to group 16SrI.

RAPID RESPONSES TO NEW PLANT DISEASES: THE USE OF GOING PUBLIC TO MONITOR THE SPREAD OF XANTHOMONAS WILT AND CONTROL NAPIER GRASS STUNT IN EAST AFRICA

ISHS IV International Symposium on Banana: International Conference on Banana and Plantain in Africa: Harnessing International Partnerships to Increase Research Impact RAPID RESPONSES TO NEW PLANT DISEASES: THE USE OF GOING PUBLIC TO MONITOR THE SPREAD OF XANTHOMONAS WILT AND CONTROL NAPIER GRASS STUNT IN EAST AFRICA

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.

Napier grass stunt: a new disease associated with a 16SrXI group phytoplasma in Kenya

Napier grass stunt: a new disease associated with a 16SrXI group phytoplasma in Kenya