M. A. Zainal Abidin

Mutagenic and inhibitory compounds produced by fungi affect detrimentally diagnosis and phylogenetic analyses

Microorganisms manufacture prolifically bioactive compounds. For example, fungi produce antibiotics and mycotoxins. However, many are difficult to identify and classify. Methods which rely on nucleic acid (DNA/RNA) are increasingly being used for this purpose where strains are grown in liquid or agar culture and often subjected to polymerase chain reaction (PCR) analyses. It has not been considered that self-produced mutagenic and inhibitory secondary metabolites (SM) affect DNA analysis of the target fungi. The most obvious mycotoxins and fungi to consider in this regard are aflatoxins (AFB) and Aspergillus, as AFB are the most mutagenic natural compounds. Many other fungi and SM are relevant and fungi act as a model for bacteria and plants. In fact, fungi repair damaged nucleic acid (NA) and are capable of removing toxins by employing transporter proteins. Nevertheless, these could be inhibited by bioactive metabolites. Mutagenic effects may involve inhibition of DNA stabilising enzymes. In addition, PCR is subject to false negative results. Samples of fungi with the genes of interest (e.g. a mycotoxin) may be categorized as negative and safe as a consequence. Internal amplification controls (IACs) will ameliorate the situation and need to become mandatory. These are conventionally NA that posses a sequence which will provide a PCR product (a) using the same primers employed for the target gene and (b) that will not coincide on the gel with the product of the target gene. Inhibitors and mutagens in cultures need to be minimized, and SM are an obvious source. This is a crucial issue in developing diagnostic and phylogenetic methods. The conclusions are (a) previous reports are compromised because IACs have not been employed in PCR and (b) mutagens and inhibitors may affect the very stability essential for NA analyses used in diagnostics and phylogenetics.

Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations.

First Report of Cabbage Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Malaysia

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 Kings 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.

In vitro production of Oncobasidium theobromae basidiospores

Sporulation and basidiospore production of Oncobasidium theobromae were successfully induced in vitro by growing the fungus initially on a nutrient rich Corticium culture medium, then on water agar. The cultures were incubated at 25 ± 2 °C and aerated with saturated moist air.

Characterisation of Pectobacterium carotovorum causing soft rot on Kalanchoe gastonis-bonnierii in Malaysia

Soft rot disease can be found worldwide on fleshy storage tissues of fruits, vegetables and ornamentals. The soft rot Pectobacterium carotovorum subsp. carotovorum (Pcc) is an important pathogen of Kalanchoe spp. and other ornamental plants. The disease occurs on crops in the field, greenhouses and during transit, resulting great economic damages. The economic importance of crop loss by soft rot bacteria varies by severity of the disease and value of the crop. A destructive disease on Kalanchoe gastonis-bonnierii was observed in commercial ornamental plant greenhouses in Cameron highland and Melaka, Malaysia in 2011. Samples suspected to be infested with Pectobacterium spp. were brought to the laboratory. In pathogenicity test, a suspension of 106 CFU/ml of strains was able to cause soft rot on leaves and stems. A 434 bp banding pattern on 1% agarose gel was produced in polymerase chain reaction (PCR) amplification of pectate lyase encoding gene (Pel gene). PCR amplification of the intergenic transcribed spacer (ITS) (16S–23S rRNA) ITS region with G1 and L1 primers produced two main bands at about 540 and 570 bp. The ITS-PCR products were digested with RsaI restriction enzyme. For discrimination of the P. carotovorum subsp. carotovorum (Pcc) from P. carotovorum subsp. odoriferum (Pco), all isolates subjected to α-methyl glucoside test. All isolates were identified as Pcc based on phenotypic and molecular methods. This is the first report of soft rot disease caused by P. carotovorum subsp. carotovorum on K. gastonis-bonnierii, in Malaysia.

Development of Microbial-Fortified Rice Straw Compost to Improve Plant Growth, Productivity, Soil Health, and Rice Blast Disease Management of Aerobic Rice

Abstract In aerobic rice cultivation systems, compost mulching and incorporation are important to rehabilitate the soil. Microbial-fortified compost is increasingly accepted as a safe approach in agro-waste management to recycling of crop residuals in agriculture soil and also to promote growth and suppress disease. This study aims to examine the stability and viability of the selected plant growth-promoting microorganisms (PGPM) in rice straw compost (RSC) over incubation period and its bio-efficacy in promoting rice (Oryza sativa) plant growth, productivity, soil health, and controlling of Pyricularia oryzae in aerobic cultivation conditions. Six selected PGPM: Pseudomonas aeruginosa (UPMP1), Corynebacterium agropyri (UPMP7), Enterobacter gergoviae (UPMP9), Bacillus amyloliquefaciens (UPMS3), Trichoderma harzianum (UPMT1), and Trichoderma virens (UPMT2) were used as a consortium of microbial inoculants to develop the microbial-fortified rice straw compost (MRSC). The MRSC was incorporated into mineral soil used for aerobic rice cultivation and its bio-efficacy was evaluated at harvest. The viability of Trichoderma spp. found stabilized at 6.78–6.00 log cfu/g and declined for all the bacterial isolates. At harvest, soil amended with MRSC significantly increased in plant height, leaf area index, 1000 grain weight, and productivity. The MRSC amended plots had significant low in rice blast disease severity with area under disease progress curve (AUDCP) of 748.22 unit/square, as compared to control (1782.67 unit/square). The physicochemical and microbiological properties of soil amended with MRSC were improved at harvest. The application of MRSC has potential to improve plant growth, productivity, rice blast disease management, and soil health of rice under aerobic cultivation systems.