Madagascar periwinkle, Catharanthus roseus (L.) G. Don, is a member of the Apocynaceae plant family that is native to Madagascar and produces dimeric terpenoid indole alkaloids that are used in the treatment of hypertension and cancer. Periwinkle as an indicator plant is highly susceptible to phytoplasmas and spiroplasma infection from different crops, and has been found to be naturally infected with spiroplasmas in Arizona, California, and the Mediterranean countries. In this study, surveys of suspected diseased periwinkles were conducted in various regions of Selangor State, Malaysia. Periwinkles showing rapid decline in the number and size of the flowers, premature abscission of buds and flowers, reduction in leaf size, chlorosis of the leaf tips and margins, general chlorosis, and stunting and dying plants were collected. These symptoms were widespread on periwinkle in this state. Diagnosis of the disease was based on symptomatology, grafting, serology (ELISA), PCR techniques, and cultivation. Tests for transmission by grafting were conducted using symptomatic periwinkle plants. Symptoms were induced on all eight graft-inoculated healthy periwinkles approximately 2 weeks after side grafting. Preliminary examination was performed by ELISA with Spiroplasma citri Saglio polyclonal antibody that was prepared against an Iranian S. citri isolate (H. Rahimian, unpublished data). Leaf extracts of all 24 symptomatic periwinkles gave positive ELISA reactions at OD405 readings ranging from 0.310 to 0.654 to the antibody against S. citri by the indirect ELISA method. Six healthy periwinkle leaves gave OD405 readings around 0.128. Total nucleic acids were extracted from 10 symptomatic and 5 asymptomatic plants (4). PCR using the ScR16F1/ScR16R1 primer pair designed to detect S. citri in carrot and P1/P7 and secA for1/rev3 primer pairs designed for identification of phytoplasmas were used to detect the causal agent (1-3). Amplification failed when the P1/P7 universal phytoplasma primer pair was used for diseased samples. However, the PCR assays resulted in products of 1,833 and 800 bp with ScR16F1/ScR16R1 and secA for1/rev3, respectively. Five of each ScR16F1/ScR16R1 and SecAfor1/SecArev3 products were cloned with the Topo TA cloning kit (Invitrogen, Carlsbad, CA), sequenced, and deposited as GenBank Accession Nos. HM015669 and FJ011099, respectively. Sequences for both genes indicated that S. citri was associated with the disease on periwinkle. ScR16F1/ScR16R1 products cloned from symptomatic periwinkles had 98% sequence identity with S. citri (GenBank Accession No. AM285316), while nucleotide sequences of SecAfor1/SecArev3 products had 88% sequence identity with S. citri GII3-3X (GenBank Accession No. AM285304). S. citri was cultivated from 10 S. citri-infected periwinkles using filtration and SP-4 media. Twenty culture tubes started to change culture medium color from red to yellow 1 month after cultivation. Helical and motile S. citri was observed in the dark-field microscope. To our knowledge, this is the first report on the presence and occurrence of S. citri in Southeast Asia and its association with lethal yellows on periwinkle in Malaysia. References: (1) J. Hodgetts et al. Int. J. Syst. Evol. Microbiol. 58:1826, 2008. (2) I.-M. Lee et al. Phytopathology 85:728, 1995. (3) I.-M. Lee et al. Plant Dis. 90:989, 2006. (4) Y.-P. Zhang et al. J. Virol. Methods. 71:45, 1998.
Bud rot disease is a damaging disease of oil palm in Colombia. The pathogen responsible for this disease is a species of oomyctes, Phytophthora palmivora which is also the causal pathogen of several tropical crop diseases such as fruit rot and stem canker of cocoa, rubber, durian and jackfruit. No outbreaks of bud rot have been reported in oil palm in Malaysia or other Southeast Asian countries, despite this particular species being present in the region. Analysis of the genomic sequences of several genetic markers; the internal transcribe spacer regions (ITS) of the ribosomal RNA gene cluster, beta-tubulin gene, translation elongation factor 1 alpha gene (EF-1α), cytochrome c oxidase subunit I & II (COXI and COXII) gene cluster along with amplified fragment length polymorphism (AFLP) analyses have been carried out to investigate the genetic diversity and variation of P. palmivora isolates from around the world and from different hosts in comparison to Colombian oil palm isolates, as one of the steps in understanding why this species of oomycetes causes devastating damage to oil palm in Latin America but not in other regions. Phylogenetic analyses of these regions showed that the Colombian oil palm isolates were not separated from Malaysian isolates. AFLP analysis and a new marker PPHPAV, targeting an unclassified hypothetical protein, was found to be able to differentiate Malaysian and Colombian isolates and showed a clear clade separations. Despite this, pathogenicity studies did not show any significant differences in the level of aggressiveness of different isolates against oil palm in glasshouse tests.
Lignification of the plant cell wall could serve as the first line of defense against pathogen attack, but the molecular mechanisms of virulence and disease between oil palm and Ganoderma boninense are poorly understood. This study presents the biochemical, histochemical, enzymology and gene expression evidences of enhanced lignin biosynthesis in young oil palm as a response to G. boninense (GBLS strain). Comparative studies with control (T1), wounded (T2) and infected (T3) oil palm plantlets showed significant accumulation of total lignin content and monolignol derivatives (syringaldehyde and vanillin). These derivatives were deposited on the epidermal cell wall of infected plants. Moreover, substantial differences were detected in the activities of enzyme and relative expressions of genes encoding phenylalanine ammonia lyase (EC 4.3.1.24), cinnamate 4-hydroxylase (EC 1.14.13.11), caffeic acid O-methyltransferase (EC 2.1.1.68) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195). These enzymes are key intermediates dedicated to the biosynthesis of lignin monomers, the guaicyl (G), syringyl (S) and ρ-hydroxyphenyl (H) subunits. Results confirmed an early, biphasic and transient positive induction of all gene intermediates, except for CAD enzyme activities. These differences were visualized by anatomical and metabolic changes in the profile of lignin in the oil palm plantlets such as low G lignin, indicating a potential mechanism for enhanced susceptibility toward G. boninense infection.
Madagascar periwinkle is an ornamental and a medicinal plant, and is also an indicator plant that is highly susceptible to phytoplasma and spiroplasma infections from different crops. Periwinkle lethal yellows, caused by Spiroplasma citri, is one of the most devastating diseases of periwinkle. The response of plants to S. citri infection is very little known at the transcriptome level. In this study, quantitative real-time PCR (RT-qPCR) was used to investigate the expression levels of four selected genes involved in defense and stress responses in naturally and experimentally Spiroplasma citri infected periwinkles. Strictosidine β-glucosidase involved in terpenoid indole alkaloids (TIAs) biosynthesis pathway showed significant upregulation in experimentally and naturally infected periwinkles. The transcript level of extensin increased in leaves of periwinkles experimentally infected by S. citri in comparison to healthy ones. A similar level of heat shock protein 90 and metallothionein expression was observed in healthy, naturally and experimentally spiroplasma-diseased periwinkles. Overexpression of Strictosidine β-glucosidase demonstrates the potential utility of this gene as a host biomarker to increase the fidelity of S. citri detection and can also be used in breeding programs to develop stable disease-resistance varieties.
Phytophthora palmivora has caused disease in many crops including oil palm in the South America region. The pathogen has had a significant economic impact on oil palm cultivation in Colombia, and therefore poses a threat to oil palm cultivation in other regions of the World, especially in Southeast Asia, the largest producer of the crop. This study aimed to look at the ability of isolates from Malaysia, Colombia, and other regions to cross-infect Malaysian oil palm, durian, and cocoa and to develop specific biomarkers and assays for identification, detection, and diagnosis of P. palmivora as a key component for the oil palm biosecurity continuum in order to contain the disease especially at the ports of entry. We have developed specific molecular biomarkers to identify and detect Phytophthora palmivora using polymerase chain reaction (PCR) and real-time loop mediated isothermal amplification (rt-LAMP) in various sample types such as soil and plants. The limit of detection (DNA template, pure culture assay) for the PCR assay is 5.94 × 10-2 ng µl-1 and for rt-LAMP is 9.28 × 10-4 ng µl-1. Diagnosis using rt-LAMP can be achieved within 30 min of incubation. In addition, PCR primer pair AV3F/AV3R developed successfully distinguished the Colombian and Malaysian P. palmivora isolates.
This study addressed the taxonomic position and group classification of a phytoplasma responsible for virescence and phyllody symptoms in naturally diseased Madagascar periwinkle plants in western Malaysia. Unique regions in the 16S rRNA gene from the Malaysian periwinkle virescence (MaPV) phytoplasma distinguished the phytoplasma from all previously described 'Candidatus Phytoplasma' species. Pairwise sequence similarity scores, calculated through alignment of full-length 16S rRNA gene sequences, revealed that the MaPV phytoplasma 16S rRNA gene shared 96.5 % or less sequence similarity with that of previously described 'Ca. Phytoplasma' species, justifying the recognition of the MaPV phytoplasma as a reference strain of a novel taxon, 'Candidatus Phytoplasma malaysianum'. The 16S rRNA gene F2nR2 fragment from the MaPV phytoplasma exhibited a distinct restriction fragment length polymorphism (RFLP) profile and the pattern similarity coefficient values were lower than 0.85 with representative phytoplasmas classified in any of the 31 previously delineated 16Sr groups; therefore, the MaPV phytoplasma was designated a member of a new 16Sr group, 16SrXXXII. Phytoplasmas affiliated with this novel taxon and the new group included diverse strains infecting periwinkle, coconut palm and oil palm in Malaysia. Three phytoplasmas were characterized as representatives of three distinct subgroups, 16SrXXXII-A, 16SrXXXII-B and 16SrXXXII-C, respectively.