Detection of coconut cadang-cadang viroid-like sequences in oil and coconut palm and other monocotyledons in the south-west Pacific

Fractionation, electroblotting and molecular hybridisation of nucleic acids extracted from tissue of African oil palm and coconut palm and some other monocotyledonous species, collected in several areas of the south-west Pacific region, demonstrated the presence of small nucleic acids with nucleotide sequences and secondary structure similar to coconut cadang-cadang viroid (CCCVd). The oil palms which contained CCCVd-related molecules showed orange leaf spots resembling those described for oil palm naturally infected with CCCVd in the Philippines, and also characteristic of a condition known as "genetic orange spotting" (GOS). We provide preliminary evidence that GOS is an infectious disorder caused by a viroid. The coconut palms did not show symptoms typical of cadang-cadang disease, but sometimes were chlorotic, stunted, or had a reduced yield. The possibility that the isolates represent variants of CCCVd is discussed. The data suggest that viroids with nucleotide sequences similar to CCCVd occur widely in palms and other monocotyledons outside the Philippines.     

Detection of Coconut cadang-cadang viroid sequences in oil and coconut palm by ribonuclease protection assay

A ribonuclease protection assay (RPA) has been developed for detecting Coconut cadang-cadang viroid (CCCVd) sequences. An RNA probe complementary to full-length CCCVd₂₄₆ was used, terminating at nucleotide 65 in the upper conserved region, and linked to a non-viroid 5' sequence, which acted as an internal control for ribonuclease activity. Extracts from CCCVd-infected coconut ( Cocos nucifera ) and African oil ( Elaeis guineensis ) palms protected three major fragments of approximately 250, 125 and 50 nt and a variable number of minor fragments. Extracts of healthy coconut palms, Potato spindle tuber viroid -infected tomato and transfer RNA did not protect the probe. The approximately 250 nt fragment is predicted to indicate the presence of monomers and dimers of circular CCCVd₂₄₆, linear CCCVd₂₄₆ with the same termini as the probe and point mutants of these forms. The origin of smaller protected fragments is discussed. RPA-detected CCCVd sequences in 13 of 18 oil palms surveyed in a commercial plantation in Malaysia. Signal intensity varied between the positive oil palms and was generally lower than in coconut palms infected with CCCVd. An infection phenotype was implied but not confirmed by the observation that in a group of 10 oil palms with orange leaf spotting, 9 contained CCCVd, whereas in a group of 8 palms without orange spotting, the viroid was detected in 4. Of four coconut palms in Sri Lanka shown by dot-blot assay to contain CCCVd-related RNA, one was shown by RPA to be positive for the CCCVd₂₄₆ sequence. RPA is therefore a robust and sensitive test for CCCVd sequences, and our results show that sequences closely related to CCCVd₂₄₆ are not confined to the Philippines.     

Zophiuma lobulata (Hemiptera: Lophopidae) causes Finschhafen disorder of coconut and oil palms

Finschhafen disorder (FD) affects coconut and oil palms in Papua New Guinea (PNG). It is characterised by yellow‐bronzing of fronds which begins at the tips and progresses towards the petiole. Although the planthopper Zophiuma lobulata (Hemiptera: Lophopidae) has been posited as a cause of FD, the basis of the relationship has not been established. Studies conducted previously on FD predate the availability of DNA‐based techniques to test for the involvement of plant pathogens such as phytoplasmas that cause yellows‐type diseases in many plant taxa and are transmitted by the order of insects to which Z. lobulata belongs. In this study, polymerase chain reaction (PCR) assays found no evidence of phytoplasmas or bacteria‐like organisms (BLOs) in tissues of coconut and oil palm symptomatic for FD and from Z. lobulata feeding on these plants. Further studies involved releasing Z. lobulata adults and nymphs onto caged, potted coconut and oil palms and onto palm fronds enclosed in mesh sleeves. In both experiments, chlorotic symptoms on the palms were observed in the presence of Z. lobulata. Insect‐free control palms did not exhibit chlorotic symptoms of FD. In the frond sleeve experiment, only the fronds where Z. lobulata fed developed chlorosis indicating that the disorder is not systemic. Unlike most yellows‐type diseases associated with Hemiptera, this study indicates that FD is because of a direct feeding effect on palms by Z. lobulata rather than transmission of a pathogen.     

Social media posts suggest that coconut rhinoceros beetle has established in the Western hemisphere

The coconut rhinoceros beetle (CRB: Oryctes rhinoceros L.) is one of the most damaging pests of coconut and oil palms in the Asia/Pacific region but has not been reported from the Western Hemisphere. Here, we report a possible establishment of CRB in Nayarit, Mexico, based on an analysis of social media reports and photographs indicating the presence of the beetle and the distinctive v-shaped notches in palm fronds caused by the beetle feeding. CRB populations are destructive once established, therefore, urgent actions are required to contain the pest and prevent damage to coconut and oil palm in the Americas.     

The Effects of Soil Water and Atmospheric Vapour Pressure Deficit on Stomatal Behaviour and Photosynthesis in the Oil Palm

The oil palm (Elaeis guineensis Jacq.) maintains a large leafarea throughout the year, but its productivity is limited bya low rate of dry matter production per unit leaf area. Stomatalclosure, at times of low soil water availability and high atmosphericvapour pressure deficit, is an important factor limiting photosynthesisand hence dry matter production. In this paper, laboratory andfield data are used to prepare a model of the relationshipsbetween net photosynthetic rate and stomatal conductance, andbetween stomatal conductance and environmental variables. Resultsshow that high atmospheric vapour pressure deficits may limitproduction even in parts of the world where oil palms are notnormally considered to suffer from water stress. The model canbe used to design and evaluate irrigation systems, and to helpquantify the potential value of oil palm genotypes with lowstomatal sensitivity to either VPD or available soil water foruse where irrigation is impractical. Key words: Elaeis guineensis Jacq., drought, irrigation, plant breeding     

Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

Background

Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.

Methodology/Principal Findings

We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.

Conclusions/Significance

We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.     

Teratorhabditis palmarum n. sp. (Nemata: Rhabditidae): An Associate of Rhynchophorus palmarum and R. cruentatus

Teratorhabditis palmarum n. sp., an associate of the palm weevils Rhynchophorus palmarum and R. cruentatus is described. Teratorhabditis palmarum was isolated from newly emerged adults and cocoons of R. palmarum from red-ring diseased coconut palms, Cocos nucifera, in Trinidad and Ecuador, and from red-ring diseased oil palms, Elaeis guineensis, in Colombia. Teratorhabditis palmarum was also associated internally with newly emerged adults of R. cruentatus from mature transplanted cabbage palmettos, Sabal palmetto, in Florida. Dauer juveniles of T. palmarum infested the genital capsule and body cavity of newly emerged adult female and male palm weevils. Adult nematodes isolated from cocoons and dauer juveniles from newly emerged palm weevils were cuhurable on bacterial lawns on several solid media. Females of T. palmarum have a single anteriorly directed ovary; vulva at 93-96% of the body length; short, hemispherical spicate tail; three or four teeth in the metastom; cuticle with distinct transverse punctations that change abruptly at the level of the procorpus to indentations of alternating size and arrangement; and eggs with cuticular sculpturing. Males have a crenate, peloderan bursa with a 2 + 5 + 3 pattern of bursal rays (7 extend to the margin of bursa); spicules linear, completely fused at the distal tip and dorsally for 50% of the total spicule length.     

Breeding for Short-stemmed Oil Palm in Nigeria: Fatty Acids, their Significance and Characteristics

The fatty acids of the progenies arising from the first back-crossfrom Elaeis guineensis and Elaeis oleifera interspecific hybridsto E. guineensis have been characterized. Fatty-acid patternsshowed striking variability which indicated that factors governingfatty-acid inheritance are polygenic. Although segregation forindividual fatty acids had no relationship with the large numberof morphological characters observed, a definite upward trendin the percentage of unsaturated fatty acids was clearly showncompared to the parent palms. The commercial and domestic advantagesof unsaturated fatty acids are discussed. Elaeis guineensis, oil palm, breeding, fatty acid content     

Stem rots of oil palm caused by <Emphasis Type="Italic">Ganoderma boninense</Emphasis>: Pathogen biology and epidemiology

Oil palm (Elaeis guineensis Jacq.) has been grown in Papua New Guinea since the early 1960s. The most important disease of oil palm in PNG is a stem rot of the palm base. This is the same disease that constitutes a major threat to sustainable oil palm production in SE Asia. Investigations into the causal pathogen have revealed that the stem rots in PNG are caused predominantly by the basidiomycete Ganoderma boninense, with a minor pathogen identified as G. tornatum G. tornatum was found to have a broad host range whereas G. boninense appears to be restricted to palms. The population structure of G. boninense was investigated using inter-fertility studies between isolates collected from basal stem rots on oil palm. Although the G. boninense field populations are predominantly comprised of distinct individuals, a number of isolates were found that share single mating alleles. This indicates that out-crossing had occurred over several generations in the resident or wild population of G. boninense prior to colonization of oil palm. No direct hereditary relationship between isolates on neighbouring diseased palms was found, although an indirect link between isolates causing upper stem rot and basal stem rot was detected.     

Microsatellite-based high density linkage map in oil palm (Elaeis guineensis Jacq.).

A microsatellite-based high-density linkage map for oil palm (Elaeis guinensis Jacq.) was constructed from a cross between two heterozygous parents, a tenera palm from the La Mé population (LM2T) and a dura palm from the Deli population (DA10D). A set of 390 simple sequence repeat (SSR) markers was developed in oil palm from microsatellite-enriched libraries and evaluated for polymorphism along with 21 coconut SSRs. A dense and genome-wide microsatellite framework as well as saturating amplified fragments length polymorphisms (AFLPs) allowed the construction of a linkage map consisting of 255 microsatellites, 688 AFLPs and the locus of the Sh gene, which controls the presence or absence of a shell in the oil palm fruit. An AFLP marker E-Agg/M-CAA132 was mapped at 4.7 cM from the Sh locus. The 944 genetic markers were distributed on 16 linkage groups (LGs) and covered 1,743 cM. Our linkage map is the first in oil palm to have 16 independent linkage groups corresponding to the plants 16 homologous chromosome pairs. It is also the only high-density linkage map with as many microsatellite markers in an Arecaceae species and represents an important step towards quantitative trait loci analysis and physical mapping in the E. guineensis species.     

Detection and differentiation of African coconut phytoplasmas: RFLP analysis of PCR-amplified 16S rDNA and DNA hybridisation

Phytoplasmas associated with lethal decline diseases of the coconut palm (Cocos nucifera) in west and east Africa were detected by the specific amplification of their 16S rRNA genes. The primers used were based on conserved mollicute-specific and coconut-phytoplasma 16S rRNA gene sequences. Phytoplasma 16S rDNA was amplified from all African decline affected palms, some periwinkle maintained phytoplasmas, but not from healthy palms, infected palms from Florida or from the Cocos spiroplasma and Acholeplasma sp. mollicutes. African phytoplasmas were also detected by DNA hybridisation using two probes from the palm lethal yellowing phytoplasma from Florida. Probes hybridised at moderate stringency to dot blots of lethal decline affected palms from Africa, indicating possible genetic relationships between different coconut phytoplasmas. RFLP analysis of rDNA fragments (length c. 1.45 kbp) detected polymorphisms, indicating that the pathogens found in west and east Africa are not identical. This provides a useful tool for further epidemiological studies of African coconut phytoplasmal diseases.     

Oil Palm Use by Adjacent Communities of Chimpanzees at Bossou and Nimba Mountains,West Africa

We investigated oil palm (Elaeis guineensis) use for feeding in 3 chimpanzee communities: Bossou and Seringbara in Guinea and Yealé in Côte d'Ivoire. Bossou was used as the benchmark for comparison. Bossou chimpanzees (Pan troglodytes verus) exhibit a wide range of oil palm targeted behaviors. We used direct observations of their two tool use, i.e., nut-cracking and pestle pounding, to establish strict and reliable criteria to ascertain the presence of comparable behaviors at the two adjacent Nimba sites. Based on monthly surveys of oil palms across the three sites, significant differences in patterns of use emerged. Bossou chimpanzees demonstrated the greatest frequency of oil palm use, while Seringbara chimpanzees, 6 km away, failed to exhibit any use and Yealé chimpanzees, 12 km away, showed all uses comparable to Bossou chimpanzees except pestle pounding and mature leaf pith-feeding. We examined the density and distribution of oil palms, tool availability for nut-cracking and pestle pounding, fruit, flower and nut availability, competition with sympatric species for fruit and nuts and the diversity of fruit species in the diet across the 3 sites. We found no clear difference in proximate environmental variables underlying observed variations in oil palm use among the 3 sites, yielding the conclusion that the differences are cultural. Assuming individual interchange between communities and the involvement of social learning in the intracommunity transmission and maintenance of oil palm uses, the result raises interesting questions about diffusion of behavior between neighboring chimpanzee communities.     

Differences in invasiveness between two cryptic species of the coconut beetle <Emphasis Type="Italic">Brontispa longissima</Emphasis> in Timor-Leste

Brontispa longissima (Coleoptera: Chrysomelidae) is a serious pest of coconut palm, and the species contains two cryptic species: the “Asian clade” is distributed over a wide area, including Asia and the Pacific region, whereas the “Pacific clade” is distributed in a limited area. Recent invasions and outbreaks have only been reported for the Asian clade, suggesting that invasive ability may differ between the clades. To reveal differences in invasiveness, we investigated the damage potential on coconut palm and range expansion of the two clades in Timor-Leste, where both clades are present. Distribution of the clades and of severely damaged trees indicated that range expansion and outbreaks have occurred for the Asian clade but not for the Pacific clade. The Asian clade attacked trees taller than 10 m, whereas the Pacific clade seldom attacked these trees. The preference for the taller trees, which are more abundant, can facilitate range expansion and outbreaks of the Asian clade. The beetle has spread through areas where coconut palms are continuously available, but have not expanded their distribution where coconut palms are separated by large gaps. This indicates that areas free of coconut palm could serve as buffer zones to prevent spread of this beetle.     

Comparative PCR analyses for the detection of the Cape St. Paul wilt disease phytoplasma in coconut palms in Ghana

Cape St. Paul Wilt Disease (CSPWD), the Ghanaian form of the lethal yellowing‐type diseases associated with phytoplasmas, is yet the major factor affecting the coconut industry in Ghana since 1932. Recently, a PCR assay based on a non‐ribosomal gene sequence was developed for the detection of the CSPWD phytoplasma in the West Region. Our study aimed at comparing the performance of the non‐ribosomal PCR to existing ribosomal PCRs and determine the best assay to use for the detection of the CSPWD phytoplasma within the three major coconut‐growing regions: Central, Western and Volta and in palms at different ages and disease stages. To determine the most affected region, 163 CSPWD‐affected coconut palms from 32 locations from the Central, Western and Volta Regions were randomly sampled. The analysis of the presence of the CSPWD phytoplasma in coconut palms of different ages showed that palms within the age brackets (1–10), (11–20) and (61–70) years were the most affected by CSPWD. The disease was most widespread in the Central, followed by the Western, then the Volta Region. A Euclidean similarity analysis of the infection rate across palm ages revealed three distinct clusters at a linkage distance of 6.5. The PCR assay based on the secA gene sequence yielded the highest number of coconut palms positive for the CSPWD phytoplasma compared to PCR assays targeting phytoplasma ribosomal genes. These results report an update on the detection of the CSPWD phytoplasma in the CSPWD‐affected coconut regions and across coconut palms representing different ages and disease stages and provide valuable information to support the CSPWD management in Ghana.     

Construction of a RFLP genetic linkage map for oil palm (Elaeis guineensis Jacq.).

We have developed a restriction fragment length polymorphism (RFLP) genetic map in oil palm (Elaeis guineensis Jacq.) for use in breeding programmes. A segregating population of 98 individuals was probed with 84 informative low copy clones (mainly PstI genomics). This yielded 103 scorable loci, of which 97 could be linked into 24 groups of two or more markers (n = 16 for oil palm), encompassing a total of 860 cM. The high level of linkage between markers (95%) suggests good genome coverage and very little segregation distortion of markers was observed. The mapping population, which was generated by the selfing of an important breeding palm (A 137/30), also segregated for the shell thickness character (Sh), enabling mapping of the gene conferring this major commercial trait. The nearest RFLP marker (pOPgSP1282) was located 9.8 cM from Sh in the mapping population and 6.6 cM away in a related (A137/30 x E80/29) smaller population (45 palms). Strategies for conversion of RFLP markers to a PCR format were explored and two important markers were shown to generate allelic variants following PCR amplification and restriction digestion of PCR products. The application of this map to oil palm breeding programmes is discussed.     

Multiple locus genome-wide association studies for important economic traits of oil palm

Palm oil has a balanced fatty acid composition and has no trans fat. As a result, its use in food has increased as food-labeling laws have changed to specify trans fat content. Increasing oil production is the main goal in oil palm breeding. Genetic mapping and genomic studies in palm trees are necessary to understand the genetic architecture of economic traits of importance for palm oil production. To help achieve this, we sampled 422 oil palms from MPOB (Malaysian Palm Oil Board)­Angola germplasm collection and measured 13 economic traits from these palms. Multi-locus genome-wide association studies (GWAS) were conducted using least absolute shrinkage and selection operator (LASSO) and genome-wide efficient mixed model analysis. We identified 19 quantitative trait loci (QTLs) for 8 traits. Of these, four Angola-specific QTLs associated with bunch components were detected on chromosomes 4, 8, and 11. These QTLs are potentially useful for introgression of desirable genes from the Angola palms to advanced breeding populations for improvement of bunch and oil yield traits. The majority of the QTLs were detected by LASSO-A, in which the p values of individual markers were calculated based on bootstrapped standard errors. Many of the detected QTLs are nearby known QTLs detected from linkage studies reported by other research groups. We also conducted genomic selection (GS) for the 13 traits and concluded that GS can be an effective tool for oil palm breeding. This is the first GWAS and GS study conducted on oil palm germplasm from Angola, and the results can be very useful in oil palm genetic studies and breeding.     

Fatal Yellowing of Oil Palms: Search for Viroids and Double-Stranded RNA

Fatal yellowing is a serious disease of still unknown origin affecting oil palms in several regions of Central and South America. In this study a search for viroids and viroid-like RNAs in oil palms was performed using two-dimensional gel electrophoresis and return gel electrophoresis of nucleic acid extracts. Although RNAs showing viroid-like gel-electrophoretic properties were detected, the presence of the known viroids was excluded by hybridization experiments using probes specific for potato spindle tuber viroid (PSTVd), coconut cadang-cadang viroid (CCCVd), or Coleus blumei viroid 1 (CbVd1). By using double-stranded RNA (dsRNA) specific monoclonal antibodies, which do not react with viroid RNA, we were able to show that oil palm RNAs, migrating like viroids are double-stranded RNA species. Since the same dsRNA pattern was found in extracts from diseased as well as from healthy oil palms, the dsRNAs can neither be part of the causative agent of fatal yellowing, nor are they associated with the disease. Their possible origin is discussed. In addition to the standard electrophoretic methods, which have been used for identification of viroids and viroid-like RNAs, we describe additional control experiments to differentiate unequivocally between circular single stranded and linear dsRNA.     

Exploring arboreal ant community composition and co-occurrence patterns in plantations of oil palm Elaeis guineensis in Borneo and Peninsular Malaysia

The oil palm, Elaeis guineensis , is a native of West Africa and is now extensively grown in south-east Asia. In 2005, the crop covered four million ha of Malaysia. We examined the arboreal ant fauna in two oil palm plantations that had been farmed with integrated pest management practices for at least 10 yr. Having cut and examined palm fronds from 595 and 503 palms in Borneo and Peninsular Malaysia respectively, we studied diversity in ant assemblages, analysed composition of ant mosaics by stepwise use of null-models, and explored the influence of exotic vs native species on community structure. Altogether we sampled 52 species of canopy ants, of which 23 species were shared between Borneo and Peninsular Malaysia. Seventeen species in Borneo and 24 species in Peninsular Malaysia accounted for 95% of all species occurrences (SOCs). Fortysix percent of all SOCs belonged to 12 tramp ant species with pan-tropical distributions. Forty percent of all SOCs were exotic ants. Six dominant species were found in Borneo, five in the Peninsula. The three most abundant species, Anoplolepis gracilipes , Oecophylla smaragdina and Technomyrmex albipes were dominant in both sites. Three dominants were invasive species that were found on more palms than the native group. Dominant tramp species coexisted with non-dominants more often and tolerated more species of non-dominant ants than native dominants. Null model analyses indicated that species were arranged in mosaics at both the Peninsular Malaysia and Borneo sites. Dominant species showed much less co-occurrence than expected by chance, pointing towards species segregation (even in presence of introduced, invasive species). Similar results were obtained for all species. Non-dominant ants were either positively associated with their dominants, or co-occurred randomly in the presence of dominant species. In contrast, ant assemblages on palms without dominants showed species segregation.     

Local and landscape drivers of arthropod diversity and decomposition processes in oil palm leaf axils

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Sex ratio and female sexual status of the coconut pest, Oryctes monoceros (Coleoptera: Dynastidae), differ in feeding galleries and pheromone-baited traps

Oryctes monoceros is a serious coconut pest, causing up to 40% damage in tropical Africa. Synthetic aggregation pheromone, ethyl 4-methyloctanoate, has been used to lure adults to traps. Traps with pheromone plus decaying palm material captured a high proportion of males. This raises the question whether individuals, which damage palms are receptive to the pheromone. We studied the sex ratio of the insects feeding on coconuts and those attracted to pheromone traps. Sixty two percent of adults from feeding galleries on living coconut palms were females. Pheromone with rotting palm material lured 43% females. To investigate the reasons for this difference, we compared the reproductive system of females lured to the odour traps or feeding in coconut galleries, or present in old rotting stems. Ninety six percent of the females trapped by pheromone had mated, and were sexually mature. In the galleries on living palms, 46% of females were immature, and 24% had not mated. In old rotting stems where eggs are laid and larvae develop, a mixture of 52% mated and 48% virgin females was found. Therefore, the pheromone together with the odour of rotting coconut stems signals a reproduction site to beetles, particularly mature females. In practice, the pheromone-baited traps will help in reducing the dissemination of gravid females, but will not affect directly the numbers of immature ones attacking palms. Our results show that when using pheromones for monitoring or controlling insects, the physiological status of the insects may have unexpected effects on the outcome.