Enhancement of Biocontrol Activities of Trichoderma Harzianum Rifai Through Protoplast Fusion

Enhancement of the biocontrol activities of Trichoderma harzianum Rifai against two soilborne pathogens, Sclerotium rolfsii and Ganoderma boninense through protoplast fusion was attempted. Mycelial cultures from three indigenous isolates from the rhizospheres of groundnut (IMI 378843), chilli (IMI 378844) and oil palm (IMI 378841) were used for the protoplast isolation and fusion studies. The result showed that the optimum release of viable protoplasts was obtained when mycelial cultures at the exponential stage was incubated for 4 h in Novozym 234 (Sigma) as the lytic enzyme at concentration of 7 mg/ml dissolved in 0.7 M NaCI and 0.6 M sorbitol. Pretreatment of mycelium v.ith 0.01 M 2-mercaptoethanol gave no Significant difference on protoplast yield of the three isolates studied. The protoplast yield was within the range of 10⁶-10⁸ protoplasts/ml and the average size of the protoplasts was 2.5-10.0 µm. Chemically induced fusion, using polyethylene glycol (pEG), among the three isolates yielded a total of 12 fusants. germinated 18 h after incubation in liquid Protoplast Regeneration Medium (PRM). When plated on solid PRM, the fusants regenerated into single colonies between 24-48 h after incubation. Of the 12 fusants obtained, five fusants showed non-parental type in isozyme analysis. They were further evaluated based on the cultural and morphological analysis, biomass growth, antagonistic activities against S. Tolfsii and G. boninense, tolerance to commonly used fungicides, and the ability to produce two extracellular lytic enzymes, B-l,3-glucanase and chitinase

Detection of oil palm root penetration by Agrobacterium-mediated transformed Ganoderma boninense, expressing green fluorescent protein

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots

Characterisation of pathogenesis-related genes and resistance gene candidates in banana (Musa acuminata) and their expression during host-pathogen interaction

Amplified chitinase gene sequence shared 99% homology with Musa acuminata class III acidic chitinase and beta-1,3-glucanase gene sequence was 100% homologous to Musa x paradisiaca beta-1,3-glucanase. Three nucleotide-binding sites and the leucine-rich repeat (NBS-LRR) type of putative RGCs and one serine/threonine kinase gene were characterised at the amino acid level. Kinase-2 (LVLDDVW) and kinase-3 (GSRIIITTRD) motifs in the nucleotide-binding domain were highly conserved in RGC2 and RGC3 and these genes belong to the non-TIR-NBS class RGCs. RGC1 was also clustered into non-TIR-NBS class RGCs; however, many residue substitutions were present in the kinase-2 and kinase-3 motifs. The sub-domain IX (LTEKSDVYSFGVVL) of serine/threonine protein kinase was highly conserved in RGC5 and it shared highest homology with PTH-2 from muskmelon. RT-PCR analysis revealed the differential expression of PR and RGC genes exhibited by different banana genotypes over sampling time. Chitinase was expressed during banana- FocR4 interaction in all three banana genotypes. However, its expression was high and constant in Rastali Mutiara during banana-FocR4 interaction and resulted in very low disease severity in FocR4 inoculated plants (2%) compared to Rastali wild-type (16%) and Jari Buaya (8%) at six weeks after inoculation. This suggests that chitinase may play an important role in disease resistance against FocR4. Besides, our study also shows that Rastali Mutiara can be a potential source of disease-resistant genes for molecular breeding of banana

Producing transgenic rice with improved traits and yield – how far have we come?

Improving rice production is of current global concern so that food security is maintained especially in developing nations where rice remains as the staple food. With the aid of molecular biology, various isolated genes conferring to abiotic, biotic and herbicide stress tolerance has been successfully transferred into rice. Attempts have also been made to enhance grain yield, nutritional characteristics, fragrance and photosynthetic capacity of rice. The success of a commercialized transgenic rice largely depends on the biosafety and environmental risks assessments as these information translates into consumers’ acceptance towards genetically modified (GM) rice. As the renowned Golden Rice has received the green light for field trial in the Philippines and Bangladesh, this would serve as a catalyst for better acceptance of GM food crops. A brief case study on the commercialization of transgenic BT rice in China will also be discussed. The review aims to bring useful insights for future endeavors in improving traits for rice through genetic engineering

Physiological and immunoblot analyses of a nitric oxide synthase (NOS)-like protein of pea (Pisum sativum L.)

Nitric oxide (NO) functions as a signal molecule in different biological processes in plants, including disease resistance. Its production is related to nitric oxide synthase (NOS). The application of chemicals that induce systemic resistance in plants did not induce NOS activity in pea, suggesting that NO functions upstream of salicylic acid (SA) in the signaling pathway of defense responses in plants. NOS activity was induced in both the incompatible and compatible interactions of pea with Ralstonia solanacearum and Pseudomonas syringae pv pisi, respectively, between 3 h to 6 h post-infiltration, indicating that NOS was involved in both resistance and disease development responses in pea. Antibodies raised against mammalian NOS did not have specificity in detecting a NOS-like protein in pea, suggesting that the pea NOS-like protein could be structurally different from mammalian NOS, and immunodetection of a plant NOS-like protein must be conducted with caution and verified with functional assays

Induction of Tolerance to Fusarium Wilt and Defense-Related Mechanisms in the Plantlets of Susceptible Berangan Banana Pre-Inoculated with Pseudomonas sp. (UPMP3) and Burkholderia sp. (UPMB3)

This study is aimed at assessing the ability of two endophytic bacteria originally isolated from healthy oil palm roots, Pseudomonas sp. (UPMP3) and Burkholderia sp. (UPMB3) to induce resistance in susceptible Berangan banana against Fusarium oxysporum race 4 (FocR4). Increased accumulation of resistance-related enzymes such as peroxidase (PO), phenylalanine ammonia lyase (PAL), lignithioglycolic acid (LTGA), and pathogenesis-related (PR) proteins (chitinase and β-1,3-glucanase) has been observed in plantlets treated with endophytic bacteria UPMP3 and UPMB3 singly or as mixture under glasshouse conditions. Pre-inoculation of banana plantlets with UPMP3 showed a significant reduction in Fusarium wilt incidence 72 d after challenged inoculation with FocR4. UPMB3 was less effective in suppressing Fusarium wilt compared to UPMP3, whereas, the mixture of both endophytes showed an intermediate effect. Based on these results, it is concluded that UPMP3 could be a promising biological control agent that can trigger resistance against Fusarium wilt in susceptible Berangan banana

Heat and hydrolytic enzymes treatment improved the Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.)

Reported Agrobacterium transformation efficiency of indica rice shoot apices varied from 0.4 to 13.8 %. In order to improve the transformation efficiency, modification of transformation protocols through heat and hydrolytic enzyme treatments on rice shoot apices were carried out. Transient expression study using reporter genes revealed that shoot apices heat treated for 3 min at 42 °C during bacterial immersion showed improved GFP (63.0 %) and GUS (42.5 %) expressions per plant as compared to standard protocol (34.0 % GFP and 36.25 % GUS). Shoot apices pre-treated with hydrolytic enzymes containing macerase, pectinase and cellulase at concentration ratio of 1:1:1 (w/v) also demonstrated high percentage of transient GFP (40.0 %) and GUS (35.0 %) expressions per plant. PCR analyses further confirmed the presence of GFP and GUS genes in the transformants. Stable expressions of GFP and GUS were also obtained in multiple shoots of regenerated shoot apices after 4 weeks of culturing in shoot proliferation media without hygromycin. In conclusion, the transformation efficiencies were improved significantly when heat (15.83 %) and hydrolytic enzymes (16.67 %) were applied as individual treatments as compared to the standard transformation method which only accounted for 5.83 %

First report of Exserohilum rostratum as pathogen of rice brown spot in Malaysia

Leaves with brown spot symptoms were sampled from rice planting areas in the Malaysian peninsula, including Kelantan, Penang, Kedah, Perak, and Selangor, over the planting season from November 2012 to December 2013. The disease was observed on the glumes and leaves of rice plants. Mature lesions were brown with a gray or whitish centre, whereas younger lesions were small, circular, and dark brown or purplish brown. Thirteen isolates were recovered from 100 diseased leaf samples that were plated on PDA medium. The mycelia were white in early stages but after 3 to 4 days, they turned gray or dark gray. Based on morphological characteristics, molecular data (polymerase chain reaction assay and nucleotide sequence analysis) and pathogenicity tests, the causal agent was identified as Exserohilum rostratum [Setosphaeria rostrata]. This is thought to be the first report of E. rostratum as pathogen of rice brown spot in Malaysia

Selection of reference genes for quantitative real-time PCR normalization in Ganoderma-infected oil palm (Elaies guineensis) seedlings

African oil palm (Elaeis guineensis) is an important oil bearing tree commercially cultivated in Malaysia. Palm oil is an important product for local consumption, provides enormous socio-economic benefits of trade and employment opportunities, and fulfilling the growing global demand for vegetable oils. The monoculture system has fostered the outbreak of basal stem rot (BSR) disease caused by the fungus Ganoderma boninense. Quantitative real-time PCR (qRT-PCR) is a widely used molecular technique to examine the infection effect on gene expression in oil palm. The selection of appropriate reference genes is vital for accurate data normalization. In this study, the expression stability of six housekeeping genes- β-actin, cyclophilin, GAPDH, MSD, NAD and ubiquitin were validated in oil palm root tissue after fungal infection. NormFinder and BestKeeper algorithms were used to cross-validate the expression stability of the candidate reference genes. MSD, NAD and ubiquitin were shown to exhibit the highest expression stability. These genes were recommended as reference genes for gene expression studies of oil palm root tissue at early fungal infection stage

The Phenylpropanoid Pathway and Lignin in Defense against Ganoderma boninense Colonized Root Tissues in Oil Palm (Elaeis guineensis Jacq.)

Basal stem rot, caused by the basidiomycete fungus, Ganoderma boninense, is an economically devastating disease in Malaysia. Our study investigated the changes in lignin content and composition along with activity and expression of the phenylpropanoid pathway enzymes and genes in oil palm root tissues during G. boninense infection. We sampled control (non-inoculated) and infected (inoculated) seedlings at seven time points [1, 2, 3, 4, 8, and 12 weeks post-inoculation (wpi)] in a randomized design. The expression profiles of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) genes were monitored at 1, 2, and 3 wpi using real-time quantitative polymerase chain reaction. Seedlings at 4, 8, and 12 wpi were screened for lignin content, lignin composition, enzyme activities (PAL, CAD, and POD), growth (weight and height), and disease severity (DS). Gene expression analysis demonstrated up-regulation of PAL, CAD, and POD genes in the infected seedlings, relative to the control seedlings at 1, 2, and 3 wpi. At 2 and 3 wpi, CAD showed highest transcript levels compared to PAL and POD. DS increased progressively throughout sampling, with 5, 34, and 69% at 4, 8, and 12 wpi, respectively. Fresh weight and height of the infected seedlings were significantly lower compared to the control seedlings at 8 and 12 wpi. Lignin content of the infected seedlings at 4 wpi was significantly higher than the control seedlings, remained elicited with no change at 8 wpi, and then collapsed with a significant reduction at 12 wpi. The nitrobenzene oxidation products of oil palm root lignin yielded both syringyl and guaiacyl monomers. Accumulation of lignin in the infected seedlings was in parallel to increased syringyl monomers, at 4 and 8 wpi. The activities of PAL and CAD enzymes in the infected seedlings at DS = 5–34% were significantly higher than the control seedlings and thereafter collapsed at DS = 69%