Coconut Lethal Yellowing Disease Research Articles

Characterization of Phytoplasmas associated with cassava (Manihot esculenta Crantz) infection in Southern and Southeastern Côte d'Ivoire

In Côte d'Ivoire, cassava is an important source of food and income for the population. However, its development is confronted with the attack of numerous pathogens such as phytoplasmas as observed in several countries in recent years. The main aim of this study is to diagnose cassava leaf infection in Côte d'Ivoire in order to contribute to sustainable production. Thus, cassava leaf samples were collected from nine growing localities. Molecular analyses required the use of the universal primer pairs for the direct PCR and specific primers for Nested PCR in order to detect the presence of phytoplasmas in the DNA extracted from the samples. Mosaic, vein banding, chlorosis, shoestring and witches' broom symptoms were observed, sometimes alone and other times in complex symptoms. The prevalence of leaf infections was over 70%, with average severity scores ranging from 4.29 to 4.74 depending on the cassava variety. Of the samples tested, 42 (19.44%) were positive with the specific primer (AkwaSR/GH813f) and 32 (18.39%) with the universal primer (R16mF2n/R16mR2), thus demonstrating the presence of phytoplasmas in both symptomatic and non-symptomatic cassava leaves. The amplicons obtained with the specific primer pair allowed the detection of the phytoplasma "Candidatus Phytoplasma palmicola" of subgroup XXII-B associated with the coconut lethal yellowing disease in Côte d'Ivoire in cassava grown in the different collection localities.

From coconut to land: changing livelihoods in Micaúne, Central Mozambique

This paper is about how people in Micaúne currently generate their livelihoods in the aftermath of the coconut economy, which collapsed in the 2000s due to the upsurge of the coconut lethal yellowing disease (CLYD) in the early 1990s. This created uncertainty among residents; however, they did not migrate in masses as documented in the literature on livelihood shocks. On the contrary, Micaúne residents have adopted a different strategy of waiting and observing, since they believe that their future lies in their land because of the potential wealth that could possibly materialise from their subsoil (anticipation). This highlights a major shift of land from a mere host for palm trees into a valuable commodity.

Tropicsafe project: Detection and management of lethal yellowing and grapevine yellows diseases in partner countries

The confirmation of the presence of 16SrXXII-B phytoplasmas associated with coconut lethal yellowing disease in Ghana and of 16SrIV in the Caribbean (Jamaica, Cuba, and Mexico) were the basis to study alternative plant species and potential insect vectors of this economically relevant disease carried out by the TROPICSAFE project started in 2017. In South Africa grapevine yellows disease associated with ‘Candidatus Phytoplasma asteris’, (subgroup 16SrI-B) and transmitted by the leafhopper Mgenia fuscovaria was studied in its main epidemiological aspects that are relevant to its management. In Chile and in Italy phytoplasmas belonging to diverse ribosomal subgroups and several alternative host plants were detected. Some insect species have been described as phytoplasma vectors or potential vectors in grapevine or in vineyard environments.

Multilocus Sequence Analysis Reveals Three Distinct Populations of "Candidatus Phytoplasma palmicola" with a Specific Geographical Distribution on the African Continent.

To sustain epidemiological studies on coconut lethal yellowing disease (CLYD), a devastating disease in Africa caused by a phytoplasma, we developed a multilocus sequence typing (MLST) scheme for "Candidatus Phytoplasma palmicola" based on eight housekeeping genes. At the continental level, eight different sequence types were identified among 132 "Candidatus Phytoplasma palmicola"-infected coconuts collected in Ghana, Nigeria, and Mozambique, where CLYD epidemics are still very active. "Candidatus Phytoplasma palmicola" appeared to be a bacterium that is subject to strong bottlenecks, reducing the fixation of positively selected beneficial mutations into the bacterial population. This phenomenon, as well as a limited plant host range, might explain the observed country-specific distribution of the eight haplotypes. As an alternative means to increase fitness, bacteria can also undergo genetic exchange; however, no evidence for such recombination events was found for "Candidatus Phytoplasma palmicola." The implications for CLYD epidemiology and prophylactic control are discussed. The usefulness of seven housekeeping genes to investigate the genetic diversity in the genus "Candidatus Phytoplasma" is underlined.IMPORTANCE Coconut is an important crop for both industry and small stakeholders in many intertropical countries. Phytoplasma-associated lethal yellowing-like diseases have become one of the major pests that limit coconut cultivation as they have emerged in different parts of the world. We developed a multilocus sequence typing scheme (MLST) for tracking epidemics of "Ca Phytoplasma palmicola," which is responsible for coconut lethal yellowing disease (CLYD) on the African continent. MLST analysis applied to diseased coconut samples collected in western and eastern African countries also showed the existence of three distinct populations of "Ca Phytoplasma palmicola" with low intrapopulation diversity. The reasons for the observed strong geographic patterns remain to be established but could result from the lethality of CLYD and the dominance of short-distance insect-mediated transmission.

Coconut lethal yellowing disease and the Oryctes monoceros beetle: a joint venture against coconut production in Mozambique

The coconut palm, an important cash and subsistence crop, is widely grown in the coastal regions of the tropics, including Mozambique. However, outbreak of an invasive and emerging coconut lethal yellowing disease (CLYD) is now a major challenge for the coconut industry and livelihood of millions of Mozambican. The observed successions of CLYD epidemics in Mozambique, suggest that other factors than plant-insect-phytoplasma triangle might be associated with the current disease trends. This study investigated the impact of Oryctes monoceros beetle on the CLYD epidemic in Mozambique, using questionnaire survey and direct observation in the fields. The results revealed that there was a significant and negative correlation between beetle and CLYD incidence. This negative correlation may be explained by beetle movement from adult CLYD-infected palms to younger palms which pose a great challenge to the replacement of palms lost due to CLYD. Management strategies must therefore stress the role of beetle larvae in palm trunks killed by CLYD and the threat of Oryces beetles to the replacement of death palm with new plantation.

Promising primers for detection of phytoplasma causing coconut lethal yellowing disease in Mozambique

Coconut is one of the main cash crop in Mozambique, which occupied the second position after Tanzania in coconut production in Africa. Coconut production was drastically affected by the occurrence of a devastating Coconut Lethal Yellowing Disease (CLYD) epidemics, which reduced significantly the coconut yields. CLYD symptoms triggered upon phytoplasma infection, i.e. premature fruit dropping, necrosis of the inflorescence and progressive yellowing of the leaves, are used to identify infected trees. However, the diagnostic based uniquely on symptoms is not conclusive to confirm infection, and needs to be confirmed by molecular methods. In this study, three previously described reference primers for phytoplasma detection were tested on infected samples collected in Mozambique. Since those primers gave incongruent results, 20 new primer pairs targeting the 16S rDNA region, were newly designed. To evaluate their performance in detecting coconut infecting phytoplasma, 108 samples were tested and selected positive samples confirmed by sequencing. Our results showed a new primer pair more accurate and reliable compared to the reference pairs for CLYD detection in Mozambique. Moreover, the new primer pair was able to detect a new putative phytoplasma variant in Mozambique. Therefore, this study makes an important contribution to CLYD phytoplasma molecular diagnostics and its causative agent, giving insights that may be applied to the study of CLYD phytoplasma infection systems.

Anagrus nedotepae sp. n. (Hymenoptera: Mymaridae), an egg parasitoid of Nedotepa curta (Hemiptera: Cicadellidae), a pest of coconut palm in West Africa.

A new Afrotropical species of Anagrus Haliday (Hymenoptera: Mymaridae), A. nedotepae Triapitsyn sp. n., is described and illustrated. The type series was reared from parasitized eggs of the leafhopper Nedotepa curta Dmitriev (Hemiptera: Cicadellidae) during a survey for its natural enemies in Côte d'Ivoire. This peculiar leafhopper, recently described from Ghana (Dmitriev 2016), lays its eggs on leaflets of coconut palm, and could be an economically important pest of this crop in West Africa, particularly because it is a suspected vector of the phytoplasma associated with the Côte d'Ivoire Lethal Yellowing disease of coconut, as characterized by Arocha-Rosete et al. (2014). Coconut lethal yellowing diseases cause severe economic losses to the very important coconut growing industry in several West African countries and elsewhere, being considered a threat of global economic and social significance (Gurr et al. 2016). Thus, the parasitoid may be of potential importance for natural biological control of this pest and urgently needs a scientific name to be used in further publications on its biology and ecology. Such studies are currently underway by the second author, who collected the type series of the new species.

English

In this study,  potential alternate hosts of the phytoplasma causing coconut lethal yellowing disease (CLYD) in Mozambique were investigated based on 16S rRNA and secA genes. The results reveal that the naturalized palm species, Elaeis guineensis and Borassus aethiopum are alternate hosts of CLYD phytoplasma in Mozambique. Based on the iPhyClassifier online software, the phytoplasma detected in B. aethiopum belongs to the 16Sr group XXII-A, which include ‘Candidatus Phytoplasma palmicola’ and ‘Candidatus Phytoplasma cocosnigeriae’. This is the first report associating ‘Candidatus Phytoplasma palmicola’ with wild naturalized palm species in the world. Key words: Alternate hosts, Borassus aethiopum, ‘Candidatus Phytoplasma palmicola’, Elaeis guineensis, Mozambique, palm lethal phytoplasma phylogeny.

Potential novel ‘CandidatusPhytoplasma pini’-related strain associated with coconut lethal yellowing in Mozambique

In this study, phytoplasmas associated with coconut lethal yellowing disease in Mozambique were characterized. For phylogenetic analysis the 16S rRNA genes were sequenced. Apart from ‘Candidatus Phytoplasma palmicola’ 16SrXXII-A, phylogenetic analysis revealed the presence of a double infection of ‘Ca. P. palmicola’ and a ‘Ca. P. pini’-related phytoplasmas. The second strain potentially represents a novel ‘Candidatus species’ and is closely related to ‘Ca. P. pini’.

High diversity, expanding populations and purifying selection in phytoplasmas causing coconut lethal yellowing in Mozambique

In this study, the putative phytoplasma species causing coconut lethal yellowing disease in Mozambique and Tanzania were characterized. The 16S rRNA and secA genes were sequenced. Phylogenetic analysis revealed that Mozambican coconut phytoplasmas belong to three different types: ‘Candidatus Phytoplasma palmicola’ 16SrXXII‐A, a second strain that was previously isolated in Tanzania and Kenya (16SrIV‐C), and a third strain that was different from all known lethal yellowing phytoplasma species. The third strain potentially represents a novel species and is closely related to pine phytoplasma. Co‐infection with ‘Ca. Phytoplasma pini’‐related and ‘Ca. Phytoplasma palmicola’ 16SrXXII‐A strains was observed. Furthermore, sequence variation in ‘Ca. Phytoplasma palmicola’ at the population level was consistent with purifying selection and population expansion.

Evaluation of coconut-citrus intercropping systems in the context of lethal yellowing disease of coconut in Ghana

Global coconut production is under the devastating threat of lethal yellowing diseases endemic in East and West Africa. The most practical solution to the disease problem lies in the development of resistant or tolerant coconut planting materials. Malayan Yellow Dwarf (MYD) crossed (x) Vanuatu Tall (VTT) coconut hybrid was identified in a resistance screening work to have moderate tolerance to the disease. Consequently, the hybrid was recommended for replanting of coconut fields devastated by lethal yellowing in Ghana. To stimulate a greater replanting effort however, there was the need to develop a more efficient coconut intercropping systems involving other economic tree crops that are capable of providing insurance against total crop failure and loss of income since the MYD x VTT coconut hybrid was only moderately tolerant to lethal yellowing. Four cropping systems involving the tolerant MYD x VTT coconut hybrid and Late Valencia sweet orange were evaluated in a randomized complete block design with three replicates. The cropping systems were: 1. Sole coconut (Coconut planted at 8.5m triangular spacing at 160 palms ha-1) 2. Sole citrus (Citrus planted at 6m square spacing at 277 trees ha-1) 3. Coconut-citrus intercrop I (Coconut planted at 9.5m triangular spacing at 128 palms ha-1 and intercropped with citrus at 100 trees ha-1 ) 4. Coconut-citrus intercrop II (Coconut planted at 10.5m triangular spacing at 105 palms ha-1 and intercropped with citrus at 80 trees ha-1). Citrus was planted at the convergence point of any two diagonal lines linked with four adjacent coconut palms. The MYD x VTT coconut hybrid planted at 9.5m triangular provided optimal spacing for citrus intercropping. The intercropping system did not hinder the optimal growth and yield of coconut or citrus. It enabled a more efficient use of land and generated higher productivity by fitting more trees (coconut/ citrus) to a unit area of land as compared with sole cropping. The costbenefit ratio of the intercropping came next to sole coconut planting. Nevertheless, intercropping enjoyed 26% of fruit income as insurance against lethal yellowing disease while sole coconut planting had no insurance cover.

Identification of potential insect vectors of the Cape Saint Paul Wilt Disease of coconut in Ghana by PCR

The vector of the phytoplasma responsible for the coconut lethal yellowing disease in West Africa is unknown to date. However, it is known that phytoplasmas are transmitted by leafhoppers and planthoppers, which are supposed to be the only ones able to inject the phytoplasma in the phloem. Whereas the presence of phytoplasma in the insect does not prove its capacity to transmit the disease. We have tested a large number of insects for the presence of phytoplamas by PCR (direct PCR and Nested PCR) using both primer pairs specific for all phytoplasmas and those specific for the coconut lethal yellowing disease phytoplasma. In effect the evidence of one or several species carrying the phytoplasma would direct us on the insects to focus on in our transmission cages trials.

Genetic variability among isolates of Coconut lethal yellowing phytoplasmas determined by Heteroduplex Mobility Assay (HMA)

Heteroduplex mobility assay (HMA) was used to determine genomic diversity among African isolates of coconut lethal yellowing phytoplasmas causing Cape St. Paul wilt disease (CSPD, Ghana), lethal disease (LD, Tanzania), and lethal yellowing (LYM, Mozambique). They were also compared to the Caribbean phytoplasma associated with coconut lethal yellowing (LY). A DNA fragment of 1850 bp covering the 16S rRNA gene and 16/23S intergenic spacer region of each isolate was amplified with primers P1 and P7 and subsequently submitted to HMA analysis for sequence variation. A PCR product amplified from GH5D (CSPD isolate) as a reference was combined with each PCR product and electrophoresed on polyacrylamide gels. Three groups of phytoplasmas associated with various coconut lethal yellowing diseases were identified by HMA. The samples from Mozambique (LYM) and Ghana (CSPD) formed one group, which was different from the second group, LD from Tanzania. These two groups were different from the third group of Caribbean isolates. This grouping was consistent with the genetic diversity described in the coconut yellowing-associated phytoplasmas detected after cloning, sequencing, and phylogenetic analyses. The HMA technique described here has the potential to provide a simple and rapid means to identify and to establish the diversity of isolates within the coconut lethal yellowing disease group.

First Report of Lethal Yellowing Group (16Sr IV) of Phytoplasmas in Vernonia cinerea in Jamaica.

Coconut lethal yellowing disease (CLY) has had a devastating effect on the coconut (Cocos nucifera L.) industry in Jamaica and Latin America. A study was conducted in Jamaica during 2005 to identify alternate hosts of the CLY phytoplasma. Since weeds are known to act as reservoir hosts of numerous pathogens, Vernonia cinerea (L.) (Asteraceae), a prevalent weed species on coconut farms island-wide, was collected from coconut farms in areas of high and low levels of CLY incidence, although none of the plants displayed disease symptoms. DNA was extracted from plant samples by the method of Dellaporta et al. (1) and analyzed by nested PCR assay employing phytoplasma universal rRNA operon primers P1/P7 (2,4) and LY16Sf/LY16-23Sr (3). DNA derived from CLY-diseased or healthy coconut palm served as positive and negative controls, respectively, in each assay. Amplification of an rDNA product of the expected size (1.7 kb) confirmed phytoplasma infections in 53 of 118 (44.9%) V. cinerea test plants. Twenty-seven of the rDNA PCR products were analyzed by digestion with restriction enducleases RsaI, MspI, MseI, TaqI, HinfI, and HhaI. The restriction fragment length polymorphism profiles obtained were similar to that observed in the CLY-infected coconut palm. V. cinerea rDNA amplicons were cloned and sequenced (in both directions) and a representative sequence was deposited in GenBank (Accession No. EU057983). Blast analysis determined this sequence to be most similar (99%) to that of CLY phytoplasma in Jamaica (Accession No. AF49807) and Florida (Accession No. AF498309). To our knowledge, this is the first report of the 16Sr IV group of phytoplasmas infecting V. cinerea. Presence of the lethal yellowing phytoplasmas in dicotyledonous plant species has important epidemiological implications concerning vector identity and ecology. Futhermore, it is now evident that weed control on coconut farms could assist in the management of CLY disease in Jamaica.

Introducing the Vector: How Coconut Lethal Yellowing Disease may have reached the Caribbean

Recent reports from Jamaica tell of an increase in the incidence of coconut lethal yellowing disease and raise the spectre of the epidemic of forty years ago. Is this just another coincidence in a series that seem to link the disease with Jamaica? The future threat is to susceptible coconut varieties in Caribbean islands from Puerto Rico to Trinidad and Latin American countries from Nicaragua to Brazil. Will the disease progress in domino fashion from country to country or will it jump some countries to reach others? To answer this, consider the possibility that the disease originated outside the Caribbean and if it did, how did it first reach Jamaica? Coconut lethal yellowing is a phytoplasma disease and, in the absence of any contrary evidence, plant pathologists believe that phytoplasma diseases are NOT carried from place to place by seeds. Rather, they ARE transmitted from plant to plant by insect vectors. So the first coconuts, taken to Jamaica in the 16th century would not have carried the disease. Since the palms became widespread through the island over the following three centuries (Harries 1980), it is unlikely that the disease was already present during that time. The same can be said everywhere else in Latin American and the Caribbean where all coconuts share a similar history of introduction and healthy establishment (Harries 2001). So it is unfortunate that some popular and scientific accounts of coconut lethal yellowing disease imply that “it originated in Jamaica” or that “it reached continental USA and Mexico from Jamaica”. The same disease occurs in Hispaniola (Haiti & Dominican Republic), the Bahamas (reputedly) and the Cayman Islands, as well as in Cuba. Cuba, by its greater size and proximity to the continent, might seem a more likely focus for the disease. What these accounts are indirectly acknowledging is that it was the publicity generated by a successful Research and Development program during the 1960s and 70s (Gowdie & Romney 1976) that has held Jamaica in the lethal yellowing spotlight. The disease has spread throughout Jamaica and Cuba, to a lesser extent in Haiti (but, exceptionally, not at all in Dominican Republic (Harries et al. 2001)), the Gulf Coast states of USA and Mexico and the Caribbean coast from Mexico to Honduras (Harries 2001). Before the disease inexorably moves to other Latin American countries, from Nicaragua to Brazil, and Caribbean islands, from Puerto Rico to Trinidad and Tobago, there may be more to be learned from Jamaica where, even now, disease activity seems to be reintensifying.

On-farm evaluation of the coconut hybrid, Malayan Yellow Dwarf x Vanuatu Tall for tolerance to the lethal yellowing disease of coconut in Ghana

The performance of the coconut hybrid, Malayan Yellow Dwarf x Vanuatu Tall (MYD x VTT) was assessed in Ghana for a period of 6-8 years. The hybrid has previously been identified as being tolerant to the devastating lethal yellowing disease known in Ghana as Cape Saint Paul Wilt Disease (CSPWD). CSPWD is the most important coconut disease in Ghana. It has destroyed approximately 11,000 ha of coconut in Ghana, causing economic hardship to thousands of people. Mortality due to CSPWD during the trial period was only 1.0%. The hybrid showed remarkable tolerance to CSPWD during the 8-year trial period. Mortality, inflorescence emergence and nut load depended largely on the level of farmers’ commitment to basic management practices. In well maintained farms, the hybrid performed two to three times better compared to farms where maintenance was poor.

First report of coconut lethal yellowing disease in Guatemala

The Atlantic coast of Guatemala runs between Belize and Honduras; two countries in which coconut lethal yellowing (LY) disease is highly active (Harrison et al., 2002). Coconut palms (Cocos nucifera, Atlantic tall ecotype) with symptoms indicative of LY (Harrison & Jones, 2003), including premature nutfall, necrosis of immature inflorescences, progressive frond yellowing and eventual death of the palm, were first observed at several sites along the coast during 2000. However, the syndrome differed from that reported for LY, as inflorescence necrosis was evident after, rather than before, frond yellowing. Tissues from trunks, young leaves or inflorescences were sampled from palms with and without symptoms at Estero Lagarto, Finca Baltimore, Santa María del Mar and Punta Manabique. Total DNA was extracted from tissues by a small-scale method and tested for phytoplasma DNA by a nested PCR using phytoplasma universal rRNA primer pair P1/P7, followed by LY group-specific primer pair LY16Sf/LY16Sr (Harrison et al., 2002). RFLP profiles obtained by digesting the nested PCR products with HinfI endonuclease (Harrison & Jones, 2003) were identical for all phytoplasma-positive Guatemalan palms and matched rDNA profiles of Honduran and Florida isolates of the LY phytoplasma included for comparison. LY phytoplasma was detected in all of the palms with symptoms (8/8), but none (0/8) of the symptomless palms tested positive. These results were confirmed later at the Centro de Investigación Científíca de Yucatán and the University of Florida using either LY group-specific or LY pathogen-specific PCR (Harrison et al., 1994). Following this initial survey, more LY-diseased coconut palms have been sighted at other locations along the Atlantic coast, but as of 2003, LY has not been seen during surveys of coconut populations along the Pacific coast of Guatemala. This is the first report of coconut lethal yellowing disease in Guatemala.

Genetic diversity in the coconut lethal yellowing disease phytoplasmas of East Africa

DNA primers, based on the ribosomal sequences of lethal yellowing-type disease (LYD) phytoplasmas, were used to analyse genetic variation within the lethal yellowing-type diseases of coconut in East Africa. Samples were collected from palms in Kenya, Mozambique and high, medium and low disease incidence areas of Tanzania. The mollicute-specific primer pair P1 and P6 amplified a 1.5 kbp product from all diseased palms and no product from symptomless palms, indicating that phytoplasmas were associated with all of these diseases. However, the Rohde forward and Rohde reverse primers (a second rRNA primer pair designed to detect East African LYD-associated phytoplasmas) only amplified products from Tanzanian and Kenyan diseased palms and not from those of Mozambique. Conversely, primers Ghana 813 and AK-SR, designed for specific detection of coconut-associated phytoplasmas in West Africa, amplified products only from the Mozambique palms, indicating that the phytoplasma associated with LYD in Mozambique is more closely related to those from West Africa. This was supported by restriction enzyme digestion of PCR products. DNA sequencing of PCR products from phytoplasmas within Tanzania revealed no detectable differences in the rDNA sequences of isolates from high, medium and low incidence areas.

The identity of a suspected vector of coconut lethal yellowing disease in Jamaica and notes on Caribovia intensa (Walker) (Homoptera: Cicadelloidea)

AbstractSpecimens of a leafhopper from Jamaica, previously thought to be Osbornellus bimarginatus (DeLong) and which is suspected of being the vector of lethal yellowing disease of coconut, were found to belong to a new species. This is described as O. dabeki sp.n. and compared with related species. Genitalia of the male of Caribovia intensa (Wlk.), hitherto unknown, is described, and this species is compared with and shown to be distinct from C. coffeacola (Doz.).