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 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.     

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.     

Phytoplasma distribution in coconut palms affected by lethal yellowing disease

Lethal yellowing (LY), the most devastating disease affecting the coconut palm in America, is caused by phytoplasmas known to be distributed in different parts of infected plants. However, no comprehensive reports exist on the phytoplasma distribution. This study refers to the detection of LY phytoplasma DNA using PCR in different coconut plant parts, throughout the development of the disease. Sample analysis of positive palms taken at different stages of disease development (either symptomatic or symptomless) showed differences in the percentage of LY detection between plant parts. Some parts showed a very high level of LY DNA (stem, young leaves, inflorescences, stem apex and root apex), low levels were found in the intermediate leaves and roots without apex, whereas no LY phytoplasma DNA was detected in mature leaves. The detection percentage of LY phytoplasma DNA was lowest in symptomless‐infected palms for all parts, except the stem, where phytoplasma accumulations were consistently detected. This pattern of detection among parts is consistent with the hypothesis that phytoplasmas move from photosynthate source tissues to sink tissues via the phloem mass flow process. The accumulations in the (lower) stem, prior to the appearance of symptoms, suggest that this part of the palm is where phytoplasmas first move from leaves after foliar feeding by vectors and in which they probably multiply and distribute to other palm parts, including roots. Embryos from infected palms were analysed by nested‐PCR and 28% of 394 embryos were positive. Phytoplasma DNA was detected in embryos from fruit on any of the fruiting bunches regardless the age, but no pattern of quantitative distribution throughout the bunch developmental stages was observed. Germination of seeds from LY‐positive symptomatic palms was 58% and from LY‐negative symptomless palms were 71%. No phytoplasma was detected in seedlings tested from both symptomatic and non‐symptomatic palms. Seedlings tested after 2 years did not develop LY symptoms or eventually died.     

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.     

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.     

Isolation ofAcholeplasma spp. from coconut palms affected by lethal yellowing disease in Jamaica

A total of 35 isolates ofAcholeplasma spp. were recovered from phloem sap and rotting tissues of lethal yellowing-diseased coconut palms. The highest isolation rate, 33% of samples from two palms, was obtained in a conventional mycoplasma medium supplemented with Tween 80; no isolates were recovered from healthy palm tissues or from uninoculated media constituents. Metabolic and serological tests on uncloned isolates showed that about two-thirds were strains ofA. axanthum and the remainder were related toA. oculi. These results strongly suggest that acholeplasmas occur in or on plant tissues either as pathogens, epiphytes, or saprophytes.     

Detection and Identification of Aster Yellows Phytoplasma Associated with Lipstick Yellow Frond Disease in Malaysia

Yellowing symptoms similar to coconut yellow decline phytoplasma disease were observed on lipstick palms (Cyrtostachys renda) in Selangor state, Malaysia. Typical symptoms were yellowing, light green fronds, gradual collapse of older fronds and decline in growth. Polymerase chain reaction assay was employed to detect phytoplasma in symptomatic lipstick palms. Extracted DNA was amplified from symptomatic lipstick palms by PCR using phytoplasma‐universal primer pair P1/P7 followed by R16F2n/R16R2. Phytoplasma presence was confirmed, and the 1250 bp products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas associated with lipstick yellow frond disease were isolates of ‘Candidatus Phytoplasma asteris’ belonging to the 16SrI group. Virtual RFLP analysis of the resulting profiles revealed that these palm‐infecting phytoplasmas belong to subgroup 16SrI‐B and a possibly new 16SrI‐subgroup. This is the first report of lipstick palm as a new host of aster yellows phytoplasma (16SrI) in Malaysia and worldwide.     

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.     

Artificial diets for rearing the coconut hispine beetle,Brontispa longissima (Coleoptera: Chrysomelidae), and their suitability to two specialist parasitoids

The coconut hispine beetle, Brontispa longissima (Gestro), is a serious invasive pest that infests young unopened fronds of coconut palms (Cocos nucifera L.) in Southeast Asia. We previously developed the first artificial diet for rearing B. longissima larvae, which contained a leaf powder of young coconut fronds. Because the fronds are required for healthy growth of coconut palms, it is necessary to reduce their use for rearing the beetles. In this study, we tested two new artificial diets for the beetle larvae, which contained the leaf powders of mature coconut leaves or orchard grass (Dactylis glomerata L.). Brontispa longissima successfully developed from hatching to adulthood on both the mature coconut leaf diet and orchard grass diet. The beetles reared on the mature coconut leaf diet and orchard grass diet developed faster than those reared on the young coconut leaf diet. Fecundity and egg hatchability of beetles did not differ among the three diet treatments. We then examined the suitability of beetle larvae or pupae reared on each diet as hosts for two specialist endoparasitoids, Asecodes hispinarum Boucek and Tetrastichus brontispae Ferriere. The survival rate from oviposition to adult emergence for A. hispinarum was 43.8% in hosts reared on a young coconut leaf diet, 77.1% on a mature coconut leaf diet, and 85.7% on an orchard grass diet. For T. brontispae, the survival rate was 70.0% in hosts reared on the young coconut leaf diet, 38.1% on the mature coconut leaf diet, and 66.7% on the orchard grass diet. Our results indicate these artificial diets can be useful for rearing B. longissima and its two parasitoids, helping to reduce the costs of mass rearing these insects.     

Further studies on a spiroplasma isolated from coconut palms in Jamaica

The pathogenicity of a spiroplasma isolated from coconut palms was tested by (1) transmission experiments to palms and other plants susceptible to infection by mycoplasmas, using the suspected vector of lethal yellowing, Myndus crudus, and vectors of the agents of other yellows diseases and (2) enzyme-linked immunosorbent assay (ELISA) to detect spiroplasma antigens in diseased palm tissues. Results of both these tests were negative and, as earlier attempts to repeat the isolations from lethal yellowing diseased palms had also been unsucessful, it was concluded that this organism was not the causal agent of lethal yellowing disease. Further analysis by serological tests and by polyacrylamide gel electrophoresis (PAGE) of spiroplasma proteins confirmed that the coconut isolates were related to members of the Spiroplasma citri serogroup but were distinct from other strains tested.     

Detection and differentiation of the coconut lethal yellowing phytoplasma in coconut‐growing villages of Grand‐Lahou,Côte d'Ivoire

Surveys for the Côte d'Ivoire lethal yellowing (CILY) phytoplasma were conducted in eight severely CILY‐affected villages of Grand‐Lahou in 2015. Leaves, inflorescences and trunk borings were collected from coconut palms showing CILY symptoms and from symptomless trees. Total DNA was extracted from these samples and tested by nested polymerase chain reaction/RFLP and sequence analysis of the 16S rRNA, ribosomal protein (rp) and the translocation protein (secA) genes. The CILY phytoplasma was detected in 82.9% of the symptom‐bearing palms collected from all the surveyed villages and from all the plant parts. Trunk borings were recommended as the most suitable plant tissue type for sampling. Results indicate that the CILY phytoplasma may have a westward spread to other coconut‐growing areas of Grand‐Lahou. CILY phytoplasma strains infecting coconut palms in the western region of Grand‐Lahou exhibited unique single nucleotide polymorphisms on the rp sequence compared to the strains from the eastern region. Moreover, single nucleotide polymorphisms on the SecA sequence distinguished the CILY phytoplasma from the Cape St. Paul Wilt Disease phytoplasma in Ghana, and the Lethal Yellowing phytoplasma in Mozambique.     

Experiments with the virus Rhabdionvirus oryctes against the coconut palm rhinoceros beetles Oryctes rhinoceros and Scapanes australis grossepunctatus in New Guinea

The virus Rhabdionvirus oryctes, originally discovered in larvae of the coconut palm dynastid Oryctes rhinoceros in Malaysia, is also pathogenic to second- and early thirdinstar larvae of Scapanes australis grossepunctatus, a dynastid pest of palms in New Guinea. Mortality of Scapanes larvae occurs within 13 to 15 days of infection. Some older third-instar larvae may be resistant. The possibility of using the virus against Scapanes in the field is discussed.     

The epizootiology of the baculovirus of the coconut palm rhinoceros beetle (Oryctes rhinoceros) in Tonga

The incidence and distribution of the baculovirus of the coconut palm rhinoceros beetle was determined in Tongatapu, Tonga, 7 years after being first introduced. Surveys showed the virus to be spread throughout the beetle population, affecting 14.6% of breeding sites and over 84% of all adult beetles taken. Counts of damaged palms and of breeding site occupancy indicated that beetle numbers had remained at low levels. It appears that the virus has the potential for long-term control of beetle populations in these habitats.     

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.     

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.     

Spatial distributions of the red palm mite, Raoiella indica (Acari: Tenuipalpidae) on coconut and their implications for development of efficient sampling plans

The red palm mite (Raoiella indica), an invasive pest of coconut, entered the Western hemisphere in 2004, then rapidly spread through the Caribbean and into Florida, USA. Developing effective sampling methods may aid in the timely detection of the pest in a new area. Studies were conducted to provide and compare intra tree spatial distribution of red palm mite populations on coconut in two different geographical areas, Trinidad and Puerto Rico, recently invaded by the mite. The middle stratum of a palm hosted significantly more mites than fronds from the upper or lower canopy and fronds from the lower stratum, on average, had significantly fewer mites than the two other strata. The mite populations did not vary within a frond. Mite densities on the top section of the pinna had significantly lower mite densities than the two other sections, which were not significantly different from each other. In order to improve future sampling plans for the red palm mite, the data was used to estimate the variance components associated with the various levels of the hierarchical sampling design. Additionally, presence-absence data were used to investigate the probability of no mites being present in a pinna section randomly chosen from a frond inhabited by mites at a certain density. Our results show that the most precise density estimate at the plantation level is to sample one pinna section per tree from as many trees as possible.     

Limits to ambulatory displacement of coconut mites in absence and presence of food-related cues

Ambulatory movement of plant-feeding mites sets limits to the distances they can cover to reach a new food source. In absence of food-related cues these limits are determined by survival, walking activity, walking path tortuosity and walking speed, whereas in presence of food the limits are also determined by the ability to orient and direct the path towards the food source location. For eriophyoid mites such limits are even more severe because they are among the smallest mites on earth, because they have only two pairs of legs and because they are very sensitive to desiccation. In this article we test how coconut mites (Aceria guerreronis Keifer) are constrained in their effective displacement by their ability to survive in absence of food (meristematic tissue under the coconut perianth) and by their ability to walk and orient in absence or presence of food-related cues. We found that the mean survival time decreased with increasing temperature and decreasing humidity. Under climatic conditions representative for the Tropics (27 °C and 75 % relative humidity) coconut mites survived on average for 11 h and covered 0.4 m, representing the effective linear displacement away from the origin. Within a period of 5 h, coconut mites collected from old fruits outside the perianth moved further away from the origin than mites collected under the perianth of young fruits. However, in the presence of food-related cues coconut mites traveled over 30 % larger distances than in absence of these cues. These results show that ambulatory movement of eriophyoid mites may well bring them to other coconuts within the same bunch and perhaps also to other bunches on the same coconut palm, but it is unlikely to help them move from palm to palm, given that palms usually do not touch each other.     

Ecological methods for adult populations of Oryctes rhinoceros (Coleoptera, Scarabaeidae)

ABSTRACT. 1. For comparing assessment methods, O.rhinoceros (L.) populations were monitored in five 30–50 ha plots in southern Luzon, Philippines. No consistent correlations were found between number of beetles caught in traps, amount of palm damage and number of breeding sites. Apparently, plots were too small to account for fast dispersal of beetles.
2. Coconut cap traps baited with ethylchrysanthemumate attracted O.rhinoceros adults searching for breeding sites. Several factors were identified influencing catches but trapping was found to be unsuitable for accurately assessing pest populations.
3. A new method which gives estimates on the monthly number of O.rhinoceros attacks on coconut palms, was tested in the Maldives. Palms are climbed about once a year and the sequence of fronds, the number and the position of beetle cuts are recorded.
4. Reduction with age of the length of three teeth on the fore tibia of O. rhinoceros adults was studied in the Philippines. The data was insufficient to obtain a clear correlation between age and length of teeth.
5. The observations indicated that in the Philippines after leaving their site of pupation, O.rhinoceros adults spend about 5 weeks feeding on coconut palms. This is followed by a period of about 7 weeks in breeding sites and, on occasion, additional visits to palms. With these estimates it was possible to relate palm damage records with numbers of feeding adults.