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First Report of Spiroplasma citri (-Induced) Associated with Periwinkle Lethal Yellows in Southeast Asia

Nejat N, Vadamalai G, Sijam K, Dickinson M

Plant Dis, 2011 Oct;95(10):1312.
PMID: 30731679 DOI: 10.1094/PDIS-03-11-0251

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with Spiroplasma citri Saglio polyclonal antibody that was prepared against an Iranian S. citri isolate (H. Rahimian, unpublished data).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.
An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm

Mohammadi MR, Vadamalai G, Joseph H

Commun. Agric. Appl. Biol. Sci., 2010;75(4):777-81.
PMID: 21534490

Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.
First Report of Two Distinct Phytoplasma Species, 'Candidatus Phytoplasma cynodontis' and 'Candidatus Phytoplasma asteris,' Simultaneously Associated with Yellow Decline of Wodyetia bifurcata (Foxtail Palm) in Malaysia

Naderali N, Nejat N, Tan YH, Vadamalai G

Plant Dis, 2013 Nov;97(11):1504.
PMID: 30708488 DOI: 10.1094/PDIS-04-13-0412-PDN

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV 'Candidatus Phytoplasma cynodontis' group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the 'Candidatus Phytoplasma asteris' 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia. References: (1) N. Nejat et al. Plant Pathol. 58:1152, 2009. (2) N. Nejat et al. Plant Pathol. J. 9:101, 2010. (3) Y. P. Zhang et al. J. Virol. Meth. 71:45, 1998. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.
Variants of Coconut cadang-cadang viroid isolated from an African oil palm (Elaies guineensis Jacq.) in Malaysia

Vadamalai G, Hanold D, Rezaian MA, Randles JW

Arch Virol, 2006 Jul;151(7):1447-56.
PMID: 16470341

Variants of Coconut cadang-cadang viroid have been identified in a plantation oil palm growing in Malaysia. Three size classes are described, comprising 297, 293, and 270 nt. Compared with the 296-nt form of coconut cadang-cadang viroid (CCCVd), all variants substituted C31 --> U in the pathogenicity domain and A175 --> C in the right-hand terminus. Other mutations and deletions accounted for the different sizes. These are the first sequences reported for variants of Coconut cadang-cadang viroid in a species other than coconut palm, and this is the first evidence that variants closely related to CCCVd occur outside the Philippines.
First Report of Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum on Cucumber in Malaysia

Nazerian E, Sijam K, Zainal Abidin MA, Vadamalai G

Plant Dis, 2011 Nov;95(11):1474.
PMID: 30731752 DOI: 10.1094/PDIS-10-10-0754

Cucumber (Cucumis sativus L.) is one of the most important vegetable fruits in Malaysia. Cucumber is principally grown in the states of Johor, Kelantan, and Perak. The broad host range Enterobacteriaceae pathogen, Pectobacterium carotovorum, can cause soft rot on stems or cucumber fruit. In Malaysia, cucumber is produced in a warm, humid climate, thus the plant is susceptible to attack by P. carotovorum at any time during production. In 2010, cucumber samples with wilted and chlorotic leaves, water-soaked lesions, and collapsed fruits were found in multiple fields. Small pieces of infected stems and fruit were immersed in 5 ml of saline solution (0.85% NaCl) for 20 min and then 50 μl of this suspension was spread onto nutrient agar (NA) and incubated at 27°C for 24 h. White-to-pale gray colonies with irregular margins were selected for analysis. For pathogenicity tests, cucumber fruits were surface sterilized by ethyl alcohol 70%, washed with sterilized distilled water, cut into small pieces, and inoculated with 20 μl of 108 CFU/ml suspensions of five representative strains. Cucumber plants were grown for 3 weeks in sterilized soil and their stems were inoculated with 20 μl of 108 CFU/ml of bacterial suspension. Inoculated samples and control (noninoculated) plants were placed in a growth chamber with 80 to 90% relative humidity at 27°C. Symptoms occurred on fruit slices and stems after 1 to 3 days and appeared the same as naturally infected samples, but the control samples remained healthy. Koch's postulates were fulfilled with the reisolation of cultures with the same characteristics as described earlier. Hypersensitivity reaction (HR) assays were done by infiltrating 108 CFU/ml of bacterial suspension into tobacco leaf epidermis and HR developed. All strains were subjected to biochemical and morphological assays, as well as molecular assessment. The strains were gram negative, facultative anaerobes, rod shaped, able to macerate potato slices and growth at 37°C; catalase positive; oxidase and phosphatase negative; able to degrade pectate; sensitive to erythromycin; negative for utilization of α-methyl glycoside, indole production, and reduction of sugars from sucrose; acid production from arabitol, sorbitol, and utilization of citrate were negative, but positive for raffinose and melibiose utilization. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment on agarose gel 1% (1). Amplification of intergenic transcribed spacer region by G1 and L1 primers gave two main bands at approximately 535 and 580 bp on agarose gel 1.5%. The ITS-PCR products were digested with RsaI restriction enzyme (3). On the basis of biochemical and morphological characteristics, PCR-based pel gene and characterization of the ITS region, and digestion of the ITS-PCR products with RsaI restriction enzyme, all isolates were identified as P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of soft rot caused by P. carotovorum subsp. carotovorum on cucumber from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.
First Report of Cabbage Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Malaysia

Nazerian E, Sijam K, Mior Ahmad ZA, Vadamalai G

Plant Dis, 2011 Apr;95(4):491.
PMID: 30743350 DOI: 10.1094/PDIS-09-10-0683

Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetables cultivated in Pahang and Kelantan, Malaysia. Pectobacterium carotovorum can cause soft rot on a wide range of crops worldwide, especially in countries with warm and humid climates such as Malaysia. Cabbage with symptoms of soft rot from commercial fields were sampled and brought to the laboratory during the winter of 2010. Disease symptoms were a gray to pale brown discoloration and expanding water-soaked lesions on leaves. Several cabbage fields producing white cultivars were investigated and 27 samples were collected. Small pieces of leaf samples were immersed in 5 ml of saline solution (0.80% NaCl) for 20 min to disperse the bacterial cells. Fifty microliters of the resulting suspension was spread on nutrient agar (NA) and King's B medium and incubated at 30°C for 48 h. Purification of cultures was repeated twice on these media. Biochemical and phenotypical tests gave these results: gram negative, rod shaped, ability to grow under liquid paraffin (facultative anaerobe); oxidase negative; phosphatase negative; positive degradation of pectate; sensitive to erythromycin; negative to Keto-methyl glucoside utilization, indole production and reduction sugars from sucrose were negative; acid production from sorbitol and arabitol was negative and from melibiose, citrate, and raffinose was positive. Hypersensitivity reaction on tobacco leaf with the injection of 106 CFU/ml of bacterial suspension for all strains was positive. Four representative strains were able to cause soft rot using cabbage slices (three replications) inoculated with a bacterial suspension at 106 CFU/ml. Inoculated cabbage slices were incubated in a moist chamber at 80% relative humidity and disease symptoms occurred after 24 h. Cabbage slices inoculated with water as a control remained healthy. The bacteria reisolated from rotted cabbage slices on NA had P. carotovorum cultural characteristics and could cause soft rot in subsequent tests. PCR amplification with Y1 and Y2 primers (1), which are specific for P. carotovorum, produced a 434-bp band with 15 strains. PCR amplification of the 16S-23S rRNA intergenic transcribed spacer region (ITS) using G1 and L1 primers gave two main bands approximately 535 and 580 bp and one faint band approximately 740 bp when electrophoresed through a 1.5% agarose gel. The ITS-PCR products were digested with RsaI restriction enzyme. According to biochemical and physiological characterictics (2), PCR-based pel gene (1), and analysis by ITS-PCR and ITS-restriction fragment length polymorphism (3), all isolates were identified as P. carotovorum subsp. carotovorum. This pathogen has been reported from Thailand, Indonesia, and Singapore with whom Malaysia shares its boundaries. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum in cabbage from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W. Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.
First Report of Lasiodiplodia theobromae Causing Stem Canker of Jatropha curcas in Malaysia

Sulaiman R, Thanarajoo SS, Kadir J, Vadamalai G

Plant Dis, 2012 May;96(5):767.
PMID: 30727556 DOI: 10.1094/PDIS-06-11-0482-PDN

Physic nut (Jatropha curcas L.) is an important biofuel crop worldwide. Although it has been reported to be resistant to pests and diseases (1), stem cankers have been observed on this plant at several locations in Peninsular Malaysia since early February 2008. Necrotic lesions on branches appear as scars with vascular discoloration in the tissue below the lesion. The affected area is brownish and sunken in appearance. Disease incidence of these symptomatic nonwoody plants can reach up to 80% in a plantation. Forty-eight samples of symptomatic branches collected from six locations (University Farm, Setiu, Gemenceh, Pulau Carey, Port Dickson, and Kuala Selangor) were surface sterilized in 10% bleach, rinsed twice with sterile distilled water, air dried on filter paper, and plated on water agar. After 4 days, fungal colonies on the agar were transferred to potato dextrose agar (PDA) and incubated at 25°C. Twenty-seven single-spore fungal cultures obtained from all locations produced white, aerial mycelium that became dull gray after a week in culture. Pycnidia from 30-day-old pure cultures produced dark brown, oval conidia that were two celled, thin walled, and oval shape with longitudinal striations. The average size of the conidia was 23.63 × 12.72 μm with a length/width ratio of 1.86. On the basis of conidial morphology, these cultures were identified as Lasiodiplodia theobromae. To confirm the identity of the isolates, the internal transcribed spacer (ITS) region was amplified with ITS1/ITS4 primers and sequenced. The sequences were deposited in GenBank (Accession Nos. HM466951, HM466953, HM466957, GU228527, HM466959, and GU219983). Sequences from the 27 isolates were 99 to 100% identical to two L. theobromae accessions in GenBank (Nos. HM008598 and HM999905). Hence, both morphological and molecular characteristics confirmed the isolates as L. theobromae. Pathogenicity tests were performed in the glasshouse with 2-month-old J. curcas seedlings. Each plant was wound inoculated by removing the bark on a branch to a depth of 2 mm with a 10-mm cork borer. Inoculation was conducted by inserting a 10-mm-diameter PDA plug of mycelium into the wound and wrapping the inoculation site with wetted, cotton wool and Parafilm. Control plants were treated with plugs of sterile PDA. Each isolate had four replicates and two controls. After 6 days of incubation, all inoculated plants produced sunken, necrotic lesions with vascular discoloration. Leaves were wilted and yellow above the point of inoculation on branches. The control plants remained symptomless. The pathogen was successfully reisolated from lesions on inoculated branches. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts and is common in tropical and subtropical regions of the world (2,3). To our knowledge, this is the first report of stem canker associated with L. theobromae on J. curcas in Malaysia. References: (1) S. Chitra and S. K. Dhyani. Curr. Sci. 91:162, 2006. (2) S. Mohali et al. For. Pathol. 35:385, 2005. (3) E. Punithalingam. Page 519 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Kew, Surrey, UK. 1976.
Fulltext Expression patterns of genes involved in the defense and stress response of Spiroplasma citri infected Madagascar Periwinkle Catharanthus roseus

Nejat N, Vadamalai G, Dickinson M

Int J Mol Sci, 2012;13(2):2301-2313.
PMID: 22408455 DOI: 10.3390/ijms13022301

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.
Effect of bioformulations on the biocontrol efficacy, microbial viability and storage stability of a consortium of biocontrol agents against Fusarium wilt of banana

Wong CKF, Saidi NB, Vadamalai G, Teh CY, Zulperi D

J Appl Microbiol, 2019 Aug;127(2):544-555.
PMID: 31077517 DOI: 10.1111/jam.14310

AIMS: This study sought to investigate the effect of bioformulation on the biocontrol efficacy, microbial viability and storage stability of a consortium of Pseudomonas aeruginosa DRB1 and Trichoderma harzianum CBF2 against Foc Tropical Race 4 (Foc-TR4).
MATERIALS AND RESULTS: Four bioformulations consisting of dry (pesta granules, talc powder and alginate beads) and liquid formulations were evaluated for their ability to control Foc-TR4, sustain microbial populations after application and maintain microbial stability during storage. All tested bioformulations reduced disease severity (DS) by more than 43·00% with pesta granules producing the highest reduction in DS by 66·67% and the lowest area under the disease progress curve value (468·75) in a glasshouse trial. Microbial populations of DRB1 and CBF2 were abundant in the rhizosphere, rhizoplane and within the roots of bananas after pesta granules application as compared to talc powder, alginate beads and liquid formulations 84 days after inoculation (DAI). The stability of both microbial populations after 180 days of storage at 4°C was the greatest in the pesta granule formulation.
CONCLUSION: The pesta granule formulation was a suitable carrier of biological control agents (BCA) without compromising biocontrol efficacy, microbial population and storage stability as compared to other bioformulations used in this study.
SIGNIFICANCE AND IMPACT OF THE STUDY: Pesta granules could be utilized to formulate BCA consortia into biofertilizers. This formulation could be further investigated for possible applications under agricultural field settings.
Fulltext Complete Genome Sequences of Oryctes rhinoceros nudivirus Strains Detected in Haplotype-G Oryctes rhinoceros from Johor, Malaysia

Anggraini E, Vadamalai G, Kong LL, Mat M, Lau WH

Microbiol Resour Announc, 2023 Mar 16;12(3):e0001923.
PMID: 36853043 DOI: 10.1128/mra.00019-23

Two members of the species Oryctes rhinoceros nudivirus (OrNV) were detected in O. rhinoceros haplotype G beetles collected from an oil palm plantation in Kluang and a wild coconut tree in Batu Pahat (Johor, Malaysia). OrNV strain Kluang comprised 125,794 bp, encoding 125 open reading frames (ORFs), while OrNV strain Batu Pahat comprised 124,925 bp, encoding 126 ORFs.
Fulltext Variants in the mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) infected with Oryctes rhinoceros nudivirus in oil palm and coconut plantations

Anggraini E, Vadamalai G, Kong LL, Mat M, Lau WH

Sci Rep, 2023 Oct 06;13(1):16850.
PMID: 37803044 DOI: 10.1038/s41598-023-43691-w

The CRB (coconut rhinoceros beetle) haplotype was classified into CRB-S and CRB-G, based on the presence of single nucleotide polymorphisms (SNPs) in the mitochondrial cox1 gene. Mitochondrial genomes (mitogenomes) are the most widely used genetic resources for molecular evolution, phylogenetics, and population genetics in relation to insects. This study presents the mitogenome CRB-G and CRB-S which were collected in Johor, Malaysia. The mitogenome of CRB-G collected from oil palm plantations in 2020 and 2021, and wild coconut palms in 2021 was 15,315 bp, 15,475 bp, and 17,275 bp, respectively. The CRB-S was discovered in coconut and oil palms in 2021, and its mitogenome was 15,484 bp and 17,142 bp, respectively. All the mitogenomes have 37 genes with more than 99% nucleotide sequence homology, except the CRB-G haplotype collected from oil palm in 2021 with 89.24% nucleotide sequence homology. The mitogenome of Johor CRBs was variable in the natural population due to its elevated mutation rate. Substitutions and indels in cox1, cox2, nad2 and atp6 genes were able to distinguish the Johor CRBs into two haplotypes. The mitogenome data generated in the present study may provide baseline information to study the infection and relationship between the two haplotypes of Johor CRB and OrNV in the field. This study is the first report on the mitogenomes of mixed haplotypes of CRB in the field.
Fulltext A Consortium of Pseudomonas aeruginosa and Trichoderma harzianum for Improving Growth and Induced Biochemical Changes in Fusarium Wilt Infected Bananas

Wong CKF, Zulperi D, Saidi NB, Vadamalai G

Trop Life Sci Res, 2021 Mar;32(1):23-45.
PMID: 33936549 DOI: 10.21315/tlsr2021.32.1.2

Fusarium wilt of banana cannot be effectively controlled by current control strategies. The most virulent form that caused major losses in the banana production is Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc-TR4). Biocontrol of Foc-TR4 using microbial antagonists offers a sustainable and eco-friendly alternative. A consortium of biocontrol agents (BCAs), Pseudomonas aeruginosa DRB1 and Trichoderma harzianum CBF2 was formulated into pesta granules, talc powder, alginate beads and liquid bioformulations. Previous study indicated bioformulations containing both BCAs successfully reduced the disease severity of Foc-TR4. To date, the biocontrol mechanism and plant growth promoting (PGP) traits of a consortium of BCAs on infected bananas have not been explored. Therefore, the study was undertaken to investigate the effect of a consortium of DRB1 and CBF2 in the growth and biochemical changes of Foc-TR4 infected bananas. Results indicated pesta granules formulation produced bananas with higher biomass (fresh weight: 388.67 g), taller plants (80.95 cm) and larger leaves (length: 39.40 cm, width: 17.70 cm) than other bioformulations. Applying bioformulations generally produced plants with higher chlorophyll (392.59 μg/g FW-699.88 μg/g FW) and carotenoid contents (81.30 μg/g FW-120.01 μg/g FW) compared to pathogen treatment (chlorophyll: 325.96 μg/g FW, carotenoid: 71.98 μg/g FW) which indicated improved vegetative growth. Bioformulation-treated plants showed higher phenolic (49.58-93.85 μg/g FW) and proline contents (54.63 μg/g FW-89.61 μg/g FW) than Foc-TR4 treatment (phenolic: 46.45 μg/g FW, proline: 28.65 μg/g FW). The malondialdehylde (MDA) content was lower in bioformulation treatments (0.49 Nm/g FW-1.19 Nm/g FW) than Foc-TR4 treatment (3.66 Nm/g FW). The biochemical changes revealed that applying bioformulations has induced host defense response by increasing phenolic and proline contents which reduced root damage caused by Foc-TR4 resulting in lower MDA content. In conclusion, applying bioformulations containing microbial consortium is a promising method to improve growth and induce significant biochemical changes in bananas leading to the suppression of Foc-TR4.
'Candidatus Phytoplasma malaysianum', a novel taxon associated with virescence and phyllody of Madagascar periwinkle (Catharanthus roseus)

Nejat N, Vadamalai G, Davis RE, Harrison NA, Sijam K, Dickinson M, et al.

Int J Syst Evol Microbiol, 2013 Feb;63(Pt 2):540-548.
PMID: 22523165 DOI: 10.1099/ijs.0.041467-0

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.
Characterization of Coconut cadang-cadang viroid variants from oil palm affected by orange spotting disease in Malaysia

Wu YH, Cheong LC, Meon S, Lau WH, Kong LL, Joseph H, et al.

Arch Virol, 2013 Jun;158(6):1407-10.
PMID: 23397332 DOI: 10.1007/s00705-013-1624-8

A 246-nt variant of Coconut cadang-cadang viroid (CCCVd) has been identified and described from oil palms with orange spotting symptoms in Malaysia. Compared with the 246-nt form of CCCVd from coconut, the oil palm variant substituted C(31)→U in the pathogenicity domain and G(70)→C in the central conserved domain. This is the first sequence reported for a 246-nt variant of CCCVd in oil palms expressing orange spotting symptoms.
Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection

Nejat N, Cahill DM, Vadamalai G, Ziemann M, Rookes J, Naderali N

Mol Genet Genomics, 2015 Oct;290(5):1899-910.
PMID: 25893418 DOI: 10.1007/s00438-015-1046-2

Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host-phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.
Molecular and biological characterization of citrus bent leaf viroid from Malaysia

Iftikhar Y, Khoo YW, Murugan T, Roslin NA, Adawiyah R, Kong LL, et al.

Mol Biol Rep, 2022 Feb;49(2):1581-1586.
PMID: 34773552 DOI: 10.1007/s11033-021-06930-9

BACKGROUND: A 328-nucleotide variant of citrus bent leaf viroid (CBLVd) was characterized by citrus varieties in Malaysia. After the first report in Malaysia, the emerging CBLVd was detected in five citrus species, namely Citrofortunella microcarpa, Citrus aurantifolia, Citrus hystrix, Citrus maxima, and Citrus sinensis.
METHODS AND RESULTS: CBLVd was detected in 23 out of 133 symptomatic samples through RT-PCR. Sequence analysis of the RT-PCR amplicons from this study showed 99-100% sequence identity to the reference CBLVd Jp isolate and CBLVd isolates reported in Malaysia. Inoculation of sap, obtained from a CBLVd positive sample, into 6-month old healthy C. microcarpa seedlings showed symptoms of slight leaf bending, reduced leaf size of matured leaves, and mild mosaic between 4 to 6 months after inoculation. Moreover, the observed symptoms of chlorosis, midvein necrosis, leaf rolling, and smalling of leaves in calamondin, C. microcarpa (Bunge) Wijnands, were not reported in earlier studies and opened a new avenue for the study of symptomology. The mechanical transmissibility of CBLVd in the inoculated seedlings was reconfirmed by RT-PCR assay and sequencing.
CONCLUSIONS: Based on the results, the sequence similarity of CBLVd isolates from different areas of Malaysia showed no significant difference among each other and the reference isolate. The CBLVd is mechanically transmissible and could produce variable symptoms in different hosts.
Fulltext Draft Genome Sequence of Bacillus tropicus Strain UPM-CREST01, Isolated from the Bulk Paddy Soil at Kampung Gajah, Perak

Mohd Azim Khan NA, Jaya Jothi S, Mohd Hata E, Parimannan S, Vadamalai G, Rajandas H

Microbiol Resour Announc, 2022 Feb 17;11(2):e0115621.
PMID: 35084217 DOI: 10.1128/mra.01156-21

We characterized the draft genome of the potentially beneficial Bacillus tropicus strain UPM-CREST01, which was isolated from the bulk soil at a paddy cultivation area in Kampung Gajah, Perak, Malaysia. The final draft assembly of 5,252,705 bp, with a G+C content of 35.23%, was found to harbor 5,368 coding sequences, including several plant-growth-promoting genes.
Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Thanarajoo SS, Kong LL, Kadir J, Lau WH, Vadamalai G

J Virol Methods, 2014 Jun;202:19-23.
PMID: 24631346 DOI: 10.1016/j.jviromet.2014.02.024

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd.
Analysis of Coconut cadang-cadang viroid variants on field samples exhibiting variation in orange spotting symptom expression and severity

Roslan ND, Sundram S, Hong LW, Ling KL, Vadamalai G

Mol Biol Rep, 2023 Nov;50(11):9699-9705.
PMID: 37676433 DOI: 10.1007/s11033-023-08771-0

BACKGROUND: Sequence variation has been attributed to symptom variations but has not been investigated in Orange Spotting-Coconut cadang-cadang viroid (OS-CCCVd) infected palms. Likewise, the relationship between Coconut cadang-cadang viroid (CCCVd) variants, Orange Spotting (OS) severity and the accumulation of the viroid in the palms have not been elucidated. This paper describes the characterization of CCCVd variants by cloning and sequencing, followed by correlation with symptom expression.
METHODS AND RESULTS: Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura × Pisifera (D × P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21 years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279.
CONCLUSION: The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.
'Candidatus Phytoplasma wodyetiae', a new taxon associated with yellow decline disease of foxtail palm (Wodyetia bifurcata) in Malaysia

Naderali N, Nejat N, Vadamalai G, Davis RE, Wei W, Harrison NA, et al.

Int J Syst Evol Microbiol, 2017 Oct;67(10):3765-3772.
PMID: 28905707 DOI: 10.1099/ijsem.0.002187

Landscape-grown foxtail palm (Wodyetia bifurcata A. K. Irvine) trees displaying symptoms of severe foliar chlorosis, stunting, general decline and mortality reminiscent of coconut yellow decline disease were observed in Bangi, Malaysia, during 2012. DNA samples from foliage tissues of 15 symptomatic palms were analysed by employing a nested PCR assay primed by phytoplasma universal ribosomal RNA operon primer pairs, P1/P7 followed by R16F2n/R2. The assay yielded amplicons of a single band of 1.25 kb from DNA samples of 11 symptomatic palms. Results from cloning and sequence analysis of the PCR-amplified 16S rRNA gene segments revealed that, in three palms, three mutually distinct phytoplasmas comprising strains related to 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma cynodontis', as well as a novel phytoplasma, were present as triple infections. The 16S rRNA gene sequence derived from the novel phytoplasma shared less than 96 % nucleotide sequence identity with that of each previously describedspecies of the provisional genus 'Ca. Phytoplasma', justifying its recognition as the reference strain of a new taxon, 'Candidatus Phytoplasma wodyetiae'. Virtual RFLP profiles of the R16F2n/R2 portion of the 16S rRNA gene and the pattern similarity coefficient value (0.74) supported the delineation of 'Ca. Phytoplasma wodyetiae' as the sole representative subgroup A member of a new phytoplasma ribosomal group, 16SrXXXVI.

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