Molecular identification and phylogenetic analysis of Pseudoperonospora cubensis isolates in Peninsula Malaysia

Thirteen isolates of Pseudoperonospora cubensis, the causal agent of downy mildew, were collected from cucurbit fields in five states of the western part of Peninsular Malaysia during its growing season between November 2008 and March 2009. The host range of these isolates was determined previously using leaf disc assay and the results indicated that there were 12 pathotypes among these isolates. The objective of this study was to analyze the 13 isolates for phylogenetic relationship using internal transcribed spacers (ITS) of ribosomal DNA (rDNA) and mitochondrial COX-II regions. A high sequence similarity among the 13 isolates and similar sequences from GenBank were detected in ITS (>99%) and COX-II (>98%) regions. Phylogenetic analysis of the 13 isolates based on Minimum Evolution method performed on ITS and COX-II regions revealed five and three groupings, respectively. However, no relationship was found between the phylogenetic groupings using both genes and pathotypes in this study

Characterization of cucumber mosaic virus (CMV) causing mosaic symptom on Catharanthus roseus (L.) G. don in Malaysia

A cucumber mosaic virus (CMV) isolate, causing leaf mosaic and distortion, malformed flowers or colourbreaking on the petals of Catharanthus roseus in Serdang, Selangor, Malaysia, was identified and designated as Malaysian periwinkle isolate (CMV-MP). The virus was spherical in shape with the size of 28.6 ± 0.48 nm in diameter with a central core. It was mechanically transmitted to various test plants which produced typical symptoms of CMV infection. The coat protein (CP) gene of the virus was amplified using reverse transcriptasepolymerase chain reaction (RT-PCR) and cloned in Escherichia coli using TOPO-TA vector. A single open reading frame of 657 nucleotides, potentially encoding for 218 amino acids was sequenced. A comparison with the CP genes of other CMV isolates indicated that CMV-MP shared 100% sequence homology to the CP gene sequence of C. roseus isolate of CMV in India. This is the first aetiology report on C. roseus in Malaysia showing natural mosaic disease symptoms supported with the nucleotide sequence analysis of the causal virus

Extracellular enzyme production during anamorphic growth in the edible mushroom, Pleurotus sajor-caju

Cultivation of mushrooms on lignocellulosic wastes represents a cost-effective organic recycling process. Pleurotus sajor-caju grown on cotton-waste produced relatively low levels of three components of the cellulase complex namely cellobiohydrolase (EC 3.2.1.91), CMCase (EC 3.2.1.4) and β-glucosidase (EC 3.2.1.21) with specific activity values of 10.0, 71.4 and 21.6U (mg protein)−1 respectively after 15days. Higher specific activity was registered in alkali-treated cotton with 15.6, 83.4 and 56.1U (mg protein)−1 respectively after 20days. Lower levels were noted on rubber-tree sawdust substrate with specific activity values of 0.28, 0.62 and 0.75U (mg protein)−1 for the respective enzymes after 28–35days growth. The maximum production of xylanase (EC 3.2.1.8) of 0.63U (mg protein)−1 occurred after 20days while a relatively higher level of the phenoloxidase enzyme, laccase (EC 1.14.18.1) of 27.4U (mg protein)−1 (maximum) was found after 35days. Laccase, the activity of which is associated with morphogenesis, increased with mycelial growth, peaked at maximum growth and thereafter decreased rapidly. This could prove important commercially in timing the end of spawn-run in preparation for initiation of fruiting

Ultrastructural features of Catharanthus roseus leaves infected with cucumber mosaic virus

Catharanthus roseus var. rosea, infected with Malaysian isolate of cucumber mosaic virus (CMV-MP), exhibited leaf mosaic, leaf deformation and malformed flowers. Electron microscopic examination of the infected leaf cells revealed significant alteration of the chloroplasts in the mesophyll cells. Large starch grains in necrotic zones and disorganized thylakoid system were the most prominent modifications observed within the chloroplasts of the infected tissues. Meanwhile, membrane-bound vesicles were detected in the vacuoles of the CMV-MP-infected leaf cells. A crystalline array of phytoferritin macromolecules was detected in the chloroplast at 40 days post-inoculation. However, neither single nor aggregate of CMV-MP particles was detected in the cytoplasm due to difficulties in differentiating them from the ribosomes. Nonetheless, structure resembling the inclusion bodies, commonly produced after virus infection, could not be found in the infected leaf cells. Similarly, structure abnormality in the nucleus or mitochondria was also not observed

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

Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability.

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp, infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. nigef) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it does not work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2

A review on bioactive compounds isolated from plants active against phytopathogenic fungi.

Plant-derived compounds are regarded as a substantial source for novel lead structures to develop medicines and biocides natural products. Concurrent with greater awareness towards the use of synthetic chemicals in agricultural practice, the application of integrated pest management programs has also increased. In recent years, there has been considerable public pressure to reduce the use of synthetic fungicides in agriculture. Although, the use of synthetic fungicides in plant disease control has been successful in improving agricultural output, several of these have been found to exhibit side-effects in the form of carcinogenicity, detrimental effects and other residual toxicities. The alternative choice therefore would be the use of botanical fungicides, which are found to be largely non-phytotoxic, systematic and easily biodegradable in nature. The present study is a summary of review literature during past decades which focused on bioactive compounds isolated from plants against plant pathogenic fungi

Evaluation of conidial viability of entomopathogenic fungi as influenced by temperature and additive

The study was conducted to evaluate the conidial viability of entomopathogenic fungi as influenced by temperature and additives. Initially five fungal isolates i.e. Metarhizium anisopliae (isolates- MPs, MaBg and MaCc1a), Beauveria bassiana (isolate- BbGc) and Paecilomyces fumosoroseus (isolate- PfPx) were screened by exposing conidia of each isolate to wet heat and oven heat stress through a series of temperature. Isolate MPs showed the best tolerance to the heat stress. The conidial germination of this isolate was 100%, when conidia were exposed at 30 to 35°C temperature for all exposure intervals. Thereafter, the effect of additive was investigated on conidial viability of the isolate MPs. A total of four commonly used components and their recommended percentage used for water-dispersible granules (WG) have passed the test. Tersperse®2700 (a dispersant), 1-naphthalene sulfonic acid, sodium salt (a wetter), lignosulfonic acid, sodium salt (a dispersant-cum-binder), sodium acetate (a disintegrant), sodium alginate and sodium glutamate (as nutritive sources as well as protectant) were selected as basic components for WG-conidia formulation as they were not harmful to MPs with germination beyond 80%, when conidia were exposed to these additives. Terwet®1004 and alginic acid failed to obtain more than 80% conidial germination, hence were excluded as ingredients of WG for causing adverse effects on conidial viability. The results indicate that the conidia of this isolate might be useful as active ingredient to produce commercial WG-conidia formulation