M. Sariah

SSRs for Marker-Assisted Selection for Blast Resistance in Rice (Oryza sativa L.)

Rice blast caused by the fungus Magnaporthe oryzae is one of the most devastating diseases of rice in nearly all rice growing areas of the world including Malaysia. To develop cultivars with resistance against different races of M. oryzae, availability of molecular markers along with marker-assisted selection strategies are essential. In this study, 11 polymorphic simple sequence repeat (SSR) markers with good fit of 1:2:1 ratio for single gene model in F2 population derived from the cross of Pongsu seribu 2 (Resistant) and Mahsuri (Susceptible) rice cultivars were analysed in 296 F3 families derived from individual F2 plants to investigate association with Pi gene conferring resistance to M. oryzae pathotype. Parents and progeny were grouped into two phenotypic classes based on their blast reactions. Chi-square test for the segregation of resistance and susceptibility in F3 generation fitted a ratio of approximately 3:1. Association of SSR markers with phenotypic trait in F3 families was identified by statistical analysis. Four SSR markers (RM413, RM5961, RM1233 and RM8225) were significantly associated with blast resistance to pathotype 7.2 of M. oryzae in rice (p ≤ 0.01). These four markers accounted for about 20% of total phenotypic variation. So, these markers were confirmed as suitable markers for use in marker-assisted selection and confirmation of blast resistance genes to develop rice cultivars with durable blast resistance in Malaysian rice breeding programmes.

Analysis of simple sequence repeat markers linked with blast disease resistance genes in a segregating population of rice (Oryza sativa).

Among 120 simple sequence repeat (SSR) markers, 23 polymorphic markers were used to identify the segregation ratio in 320 individuals of an F(2) rice population derived from Pongsu Seribu 2, a resistant variety, and Mahsuri, a susceptible rice cultivar. For phenotypic study, the most virulent blast (Magnaporthe oryzae) pathotype, P7.2, was used in screening of F(2) population in order to understand the inheritance of blast resistance as well as linkage with SSR markers. Only 11 markers showed a good fit to the expected segregation ratio (1:2:1) for the single gene model (d.f. = 1.0, P < 0.05) in chi-square (χ(2)) analyses. In the phenotypic data analysis, the F(2) population segregated in a 3:1 (R:S) ratio for resistant and susceptible plants, respectively. Therefore, resistance to blast pathotype P7.2 in Pongsu Seribu 2 is most likely controlled by a single nuclear gene. The plants from F(2) lines that showed resistance to blast pathotype P7.2 were linked to six alleles of SSR markers, RM168 (116 bp), RM8225 (221 bp), RM1233 (175 bp), RM6836 (240 bp), RM5961 (129 bp), and RM413 (79 bp). These diagnostic markers could be used in marker assisted selection programs to develop a durable blast resistant variety.

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.

Assessment of plant secondary metabolites in oil palm seedlings after being treated with calcium, copper ions and salicylic acid

The effect of calcium, copper ions and salicylic acid (SA) amendment on the incidence of basal stem rot and activity of secondary metabolites in oil palm seedlings were investigated in glasshouse study. Disease incidence (DI) in positive control (T8) was 75% at nine months after inoculation (9 MAI). However, weekly pre-immunisation with Ca2+ + Cu2+ + SA prior to inoculation significantly suppressed DI and delayed disease onset as noted in T7. In the present study, the lowest %DI was observed in T7 (15%) followed by T1, T5, T6, T3, T4 and T2. The Ca2+, Cu2+ and SA amendments were resulted in earlier and higher accumulation of plant secondary metabolites as noted in leaves, stems and root tissues in response to invasion by Ganoderma boninense. High total phenolic content concentration was detected in T7 (leaf: 233.38 ± 0.12 mg/g; stem: 132.78 ± 0.04 mg/g and root: 86.98 ± 0.28 mg/g). Similar trend was obtained in peroxidase activity, total lignin content and hydrogen peroxide scavenging activity. These results suggested that it could be due to the accumulation of phenolics, peroxidase activities, lignin content and hydrogen peroxide scavenging activities in oil palm seedling tissues which might have collectively contributed to induce resistance against G. boninense.

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.

Characterization of Malaysian Pectobacterium spp. from vegetables using biochemical, molecular and phylogenetic methods

In August 2011, vegetable crops showing symptoms of maceration and water soaked lesions on their tuber, leaf, and fruit were collected from four major vegetable growing states in Malaysia including Pahang, Johor, Melaka and Selangor. The majority of the causal organisms isolated from infected tissues (52 strains) were identified as Pectobacterium spp. based on PCR amplification of the pectate lyase (pel) gene and amplification of the 16S-23S rRNA (ITS) with G1 and L1 primers. Physiological and biochemical assays divided Malaysian Pectobacterium species into two main groups: Pectobacterium wasabiae and Pectobacterium carotovorum subsp carotovorum. Partial sequence of PCR product from reaction of putative Pectobacterium spp. with 16S rRNA confirmed the results obtained from physiological and biochemical assays used for identification of the bacterium. Application of specific primers such as Eca1F/Eca2r, Br1f/L1r, EXPCCF/EXPCCR, and also ITS-PCR following by RFLP by restriction enzyme (RsaI) successfully differentiated Malaysian P. wasabiae and P. carotovorum subsp carotovorum isolates from other species and subspecies of Pectobacterium. Phylogenetic analysis of Malaysian isolates with housekeeping genes (mdh, gapA) grouped Malaysian P. carotovorum subsp carotovorum and P. wasabiae in the same cluster with P. carotovorum subsp carotovorum (Ecc380) and P. wasabiae (SCRI488) respectively.

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.

First report of soft rot disease caused by Pectobacterium wasabiae on sweet potato, tomato, and eggplant in Malaysia.

In August 2011, sweet potato (Ipomoea batatas), tomato (Solanum lycopersicum), and eggplant (S. melongena) crops from major growing areas of the Cameron highlands and Johor state in Malaysia were affected by a soft rot disease. Disease incidence exceeded 80, 75, and 65% in severely infected fields and greenhouses of sweet potato, tomato, and eggplant, respectively. The disease was characterized by dark and small water-soaked lesions or soft rot symptoms on sweet potato tubers, tomato stems, and eggplant fruits. In addition, extensive discoloration of vascular tissues, stem hollowness, and water-soaked, soft, dark green lesions that turned brown with age were observed on the stem of tomato and eggplant. A survey was performed in these growing areas and 22 isolates of the pathogen were obtained from sweet potato (12 isolates), tomato (6 isolates), and eggplant (4 isolates) on nutrient agar (NA) and eosin methylene blue (EMB) (4). The cultures were incubated at 27°C for 2 days and colonies that were emerald green on EMB or white to gray on NA were selected for further studies. All bacterial cultures isolated from the survey exhibited pectolytic ability on potato slices. These bacterial isolates were gram negative; rod shaped; N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG positive; and were also positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. They were negative for indol production, phosphatase activity, reducing substances from sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-mathyl-D-glocoside, and D-arabitol. The bacteria did not grow on NA at 37°C. Based on these biochemical and morphological assays, the pathogen was identified as Pectobacterium wasabiae (2). In addition, DNA was extracted and PCR assay with two primers (16SF1 and 16SR1) was performed (4). Partial sequences of 16S rRNA (GenBank Accession Nos. JQ665714, JX494234, and JX513960) of sweet potato, tomato, and eggplant, respectively, exhibited a 99% identity with P. wasabiae strain SR91 (NR_026047 and NR_026047.1). A pathogenicity assay was carried out on sweet potato tubers (cv. Oren), tomato stems (cv. 152177-A), and eggplant fruits (cv. 125066x) with 4 randomly representative isolates obtained from each crop. Sweet potato tubers, tomato stems, and eggplant fruits (4 replications) were sanitized in 70% ethyl alcohol for 30 s, washed and rinsed in sterile distilled water, and needle punctured with a bacterial suspension at a concentration of 108 CFU/ml. Inoculated tubers, stems, and fruits were incubated in a moist chamber at 90 to 100% RH for 72 h at 25°C when lesions were measured. All inoculated tubers, stems, and fruits exhibited soft rot symptoms after 72 h similar to those observed in the fields and greenhouses and the same bacteria were consistently reisolated. Symptoms were not observed on controls. The pathogenicty test was repeated with similar results. P. wasabiae have been previously reported to cause soft rot on Japanese horseradish (3), and aerial stem rot on potato in New Zealand (4), the U.S. (2), and Iran (1). To our knowledge, this is the first report of sweet potato, tomato, and eggplant soft rot caused by P. wasabiae in Malaysia. References: (1) S. Baghaee-Ravari et al. Eur. J. Plant Pathol. 129:413, 2011. (2) S. De Boer and A. Kelman. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. N. Schaad et al., eds. APS Press, St. Paul, 2001. (3) M. Goto et al. Int. J. Syst. Bacteriol. 37:130, 1987. (4) A. R. Pitman et al. Eur. J. Plant Pathol. 126:423, 2010.

Effect of cold-water irrigation on bacterial wilt pathogen of tomato

Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating bacterial diseases of plants worldwide. Management of bacterial wilt in tomato and other crops has been difficult, and so novel but easily implemented control methods are being sought. To evaluate the effect of cold-water irrigation on bacterial wilt of tomato, four treatments were used in which CF (chemically fertilized) soil and CF + FYM (chemical fertilizer + farmyard manure [FYM]) soil were inoculated with a bacterial suspension (R. solanacearum strain YU1Rif43) at 106 colony forming units (CFU) g−1 soil. Tomato seedlings were grown in Agri-pots in a plant growth chamber. The soil was irrigated with water that was kept at the same temperature in each treatment: 4, 10, 20, or 30°C. Incidence and severity of wilt, counting of the colonies of the culturable population of pathogen, and dry-mass and height of the plants were examined. After 45 days and in both kinds of soil, most of the plants had wilted in soil irrigated at 30°C. Wilt incidence was substantially reduced when transplanted seedlings were irrigated at lower temperatures (4–20°C). Survival of R. solanacearum was also reduced after being irrigated with water at lower temperatures, indicating that the reduced incidence of wilt was linked to reduced survival of the pathogen. Dry-mass and plant height were slightly higher under control conditions than in soils irrigated at lower temperatures. This study suggests that cold-water irrigation could significantly reduce bacterial wilt of tomato and have an adverse effect on survival of the wilt pathogen.