The possibility of controlling Fusarium wilt--caused by Fusarium oxysporum sp. cubensec (race 4)--was investigated by genetic engineering of banana plants for constitutive expression of rice thaumatin-like protein (tlp) gene. Transgene was introduced to cauliflower-like bodies' cluster, induced from meristemic parts of male inflorescences, using particle bombardment with plasmid carrying a rice tlp gene driving by the CaMV 35S promoter. Hygromycin B was used as the selection reagent. The presence and integration of rice tlp gene in genomic DNA confirmed by PCR and Southern blot analyses. RT-PCR revealed the expression of transgene in leaf and root tissues in transformants. Bioassay of transgenic banana plants challenged with Fusarium wilt pathogen showed that expression of TLP enhanced resistance to F. oxysporum sp. cubensec (race 4) compared to control plants.
Cosmos caudatus Kunth. (Asteraceae), commonly known as ulam raja, is widely grown as an herbal aromatic shrub. In Malaysia, its young leaves are popularly eaten raw as salad with other greens and have been reported to possess extremely high antioxidant properties, which may be partly responsible for some of its believed medicinal functions. In early 2010, a suspected powdery mildew was observed on ulam raja plants at the Agricultural Park of Universiti Putra Malaysia. Initially, individual, white, superficial colonies were small and almost circular. Later, they enlarged and coalesced to cover the whole abaxial leaf surface. With development of the disease, all green parts (leaves, stems, and petioles) became covered with a continuous mat of mildew, giving a dusty appearance. Newly emerged leaves rapidly became infected. Diseased leaves ultimately senesced and dried up, making them aesthetically unattractive and unmarketable. The pathogen produced conidia in short chains (four to six conidia) on erect conidiophores. Conidiophores were unbranched, cylindrical, 125 to 240 μm long, with a slightly swollen foot cell. Individual conidia were hyaline, ellipsoid, and 25 to 30 (27.5) × 15 to 20 (17.5) μm with fibrosin inclusions. Morphological descriptions were consistent with those described for Sphaerotheca fuliginea or S. fusca, which has lately been reclassified as Podosphaera fusca (1). From extracted genomic DNA of P. fusca UPM UR1, the internal transcribed spacer (ITS) region was amplified with ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). A BLAST search of GenBank with an ITS rDNA sequence of this fungus (GenBank Accession No. HQ589357) showed a maximum identity of 98% to the sequences of two P. fusca isolates (GenBank Accession Nos. AB525915.1 and AB525914.1). To satisfy Koch's postulates, the pathogenicity of fungal strain UPM UR1 was verified on 4-week-old plants. Inoculation was carried out by gently rubbing infected leaves onto healthy plants of C. caudatus. Ten pots of inoculated plants were kept under a plastic humid chamber and 10 pots of noninoculated plants, placed under another chamber, served as controls. After 48 h, the plants were then placed under natural conditions (25 to 28°C). Powdery mildew symptoms, similar to those on diseased field plants, appeared after 7 days on all inoculated plants. The white, superficial colonies enlarged and merged to cover large areas within 2 weeks. The infected leaf tissues became necrotic 6 to 8 days after the appearance of the first symptoms. Sporulation of P. fusca was observed on all infected leaves and stems. No symptoms were seen on the control plants. To our knowledge, this is the first report of P. fusca causing powdery mildew on C. caudatus in Malaysia. This pathogen has also been reported previously to be economically important on a number of other hosts. With ulam raja plants, more attention should be given to prevention and control measures to help manage this disease. Reference: (1) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000.
Basal stem rot of oil palm caused by Ganoderma boninense is of major economic importance. Observations of the low incidence of disease due to Ganoderma species in natural stands, suggest that the disease is kept under control by some biological means. Trichoderma spp. are saprophytic fungi with high antagonistic activities against soil-borne pathogens. However, their abundance and distribution are soil and crop specific. Trichoderma species have been found to be concentrated in the A1 (0-30 cm) and Be soil horizons (30-60 cm), although the abundance of Trichoderma was not significantly different between the oil palm and non-oil palm ecosystems. Characterisation of Trichoderma isolates based on cultural, morphological and DNA polymorphism showed that T. harzianum, T. virens, T. koningii and T. longibrachiatum made up 72, 14, 10 and 4% of the total Trichoderma isolates isolated. As Trichoderma species are present in the oil palm ecosystem, but at lower numbers and in locations different from those desired, soil augmentation with antagonistic Trichoderma spp. can be developed as a strategy towards integrated management of basal stem rot of oil palm.
Ergosterol is the main component of the fungal membrane and is not found in plants or other microbial cells. Therefore, it can be a useful biomarker for the quantification of fungal biomass. We are now reporting the first isolation and characterisation of ergosterol from the mycelium of G. boninense. The ergosterol structure was detected by Thin Liquid Chromatography (TLC) and Ultra Performance Liquid Chromatography (UPLC) and confirmed with Gas Chromatography coupled with Mass Spectrometry (GCMS) and Nuclear Magnetic Resonance (NMR) analysis.
In December 2008, infected leaves of Trichosanthes cucumerina were observed on commercial cucurbit farms located in Pontian, Johor (south of West Malaysia). Bright yellow and small necrotic lesions were observed on the adaxial surface of the leaves, whereas sporangiophores were observed on pale yellowish brown-to-brown lesions on the abaxial surface. The length and width of the sporangia ranged from 19 to 36 μm (28.6) and 11 to 23 μm (17.6), respectively. The length of the sporangiophores ranged from 310 to 450 μm, with an average length of 380 μm. The pathogen was identified as Pseudoperonospora cubensis on the basis of the morphological criteria described by Palti and Cohen (2). To confirm the morphological findings, DNA was extracted from symptomatic tissue and the internal transcribed spacer (ITS) region was PCR amplified using primers ITS5-P2 and ITS4 (3). The appropriate-sized amplicon was gel excised and column purified and then submitted for direct sequencing. The resulting 802 bp amplified ITS region was 100% identical to published P. cubensis sequences (GenBank Accession Nos. EU876603, EU876584, and AY198306). This sequence was deposited with NCBI GenBank under the Accession No. GU233293. In this study, pathogenicity tests were conducted using detached leaf disc assays (1) and a P. cubensis isolate obtained from T. cucumerina. For this purpose, leaf discs were excised from 6- to 8-week-old leaves of T. cucumerina using a 20-mm cork borer. Five leaf discs were placed with their abaxial surface facing upward on moist filter paper in petri dishes. Each of four leaf discs was inoculated with four 10-μl droplets of a 1 × 105 per ml sporangial suspension, whereas the fifth disc was inoculated with water droplets and served as a control. Three replications were completed. The leaf discs were placed in darkness at 14 ± 2°C for 24 h and subsequently incubated with a 12-h photoperiod. After 10 days, sporulation was observed on the sporangia-inoculated leaf discs with similar morphological features to the initial field samples. To our knowledge, this is the first report of P. cubensis causing downy mildew of T. cucumerina in Malaysia. References: (1) A. Lebeda and M. P. Widrlechner. J. Plant Dis. Prot. 110:337, 2003. (2) J. Palti and Y. Cohen. Phytoparasitica 8:109, 1980. (3) H. Voglmayr and O. Constantinescu. Mycol. Res. 112:487, 2008.
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.
Use of phosphate-solubilizing bacteria (PSB) as inoculants has concurrently increased phosphorous uptake in plants and improved yields in several crop species. The ability of PSB to improve growth of aerobic rice (Oryza sativa L.) through enhanced phosphorus (P) uptake from Christmas island rock phosphate (RP) was studied in glasshouse experiments. Two isolated PSB strains; Bacillus spp. PSB9 and PSB16, were evaluated with RP treatments at 0, 30 and 60 kg ha(-1). Surface sterilized seeds of aerobic rice were planted in plastic pots containing 3 kg soil and the effect of treatments incorporated at planting were observed over 60 days of growth. The isolated PSB strains (PSB9 and PSB16) solubilized significantly high amounts of P (20.05-24.08 mg kg(-1)) compared to non-inoculated (19-23.10 mg kg(-1)) treatments. Significantly higher P solubilization (24.08 mg kg(-1)) and plant P uptake (5.31 mg plant(-1)) was observed with the PSB16 strain at the highest P level of 60 kg ha(-1). The higher amounts of soluble P in the soil solution increased P uptake in plants and resulted in higher plant biomass (21.48 g plant(-1)). PSB strains also increased plant height (80 cm) and improved root morphology in aerobic rice. The results showed that inoculation of aerobic rice with PSB improved phosphate solubilizing activity of incorporated RP.
Rice straw is produced as a by-product from rice cultivation, which is composed largely of lignocellulosic materials amenable to general biodegradation. Lignocellulolytic actinobacteria can be used as a potential agent for rapid composting of bulky rice straw. Twenty-five actinobacteria isolates were isolated from various in situ and in vitro rice straw compost sources. Isolates A2, A4, A7, A9 and A24 were selected through enzymatic degradation of starch, cellulose and lignin followed by the screening for their adaptability on rice straw powder amended media. The best adapted isolate (A7) was identified as Micromonospora carbonacea. It was able to degrade cellulose, hemicelluloses and carbon significantly (P ≤ 0.05) over the control. C/N ratio was reduced to 18.1 from an initial value of 29.3 in 6 weeks of composting thus having the potential to be used in large scale composting of rice straw.
Symptoms of water-soaked lesions and soft rot were first observed in June 2011 on bell pepper fruits (Capsicum annuum cv. Annuum) in the two main regions of pepper production in Malaysia (Cameron Highlands and Johor State). Economic losses exceeded 40% in severely infected fields and greenhouses with the estimated disease incidence of 70%. In pepper fruits damaged by insects, sunscald, or other factors, symptoms initially appeared in the peduncle and calyx tissues and entire fruits were turned into watery masses within 2 to 6 days. Fruits infected in the field tended to collapse and hang on the plant. When the contents leaked out, the outer skin of the fruit dried and remained attached to the plant. Field-grown transplants and infected soil were identified as probable sources of inocula. A total of 50 attached fruits were collected from 10 pepper fields and greenhouses located in the two growing regions. Tissue from the margins of water-soaked lesions was surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto nutrient agar (NA) and eosin methylene blue agar (EMB) media (3). A similar bacterium was isolated from all samples. After 2 days, white to creamy bacterial colonies on NA and emerald green colonies on EMB developed. Five independent strains were subjected to further biochemical, molecular, and pathogenicity tests. Bacterial strains were gram-negative, motile rods, grew at 37°C, were facultatively anaerobic, oxidase-negative, phosphatase-negative, and catalase-positive. They degraded pectate, were sensitive to erythromycin, did not utilize Keto-methyl glucoside, were indole production-negative, and reduced sugars from sucrose (3). Acid production was negative from sorbitol and arabitol, but positive from melibiose and citrate. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment (2). Amplification of the intergenic transcribed spacer (ITS) region by G1 and L1 primers (4) gave two amplicons ca. 550 and 580 bp long. The expected amplicon was not produced with any of the strains using primers Br1f/L1r and Eca1f/Eca2r (1), whereas a 550-bp PCR product, typical of Pectobacterium carotovorum subsp. carotovorum, was obtained with primers EXPCCF and EXPCCR (1). Based on biochemical and molecular characteristics, and analysis of PCR-RFLP of 16S-ITS-23R rRNA genes using Rsa I enzyme (4), all five bacterial strains were identified as P. carotovorum subsp. carotovorum. BLAST analysis of the 16S rRNA sequence (GenBank Accession No KC189032) showed 100% identity to the 16S rRNA of P. carotovorum subsp. carotovorum strain PPC192. For pathogenicity tests, four mature pepper fruits of cv. Annuum were inoculated by injecting 10 μl of a bacterial suspension (108 CFU/ml) into pericarps and the fruits were incubated in a moist chamber at 80 to 90% relative humidity and 30°C. After 72 h, water-soaked lesions similar to those observed in the fields and greenhouses were observed and bacteria with the same characteristics were consistently reisolated, thereby fulfilling Koch's postulates. Symptoms were not observed on water-inoculated controls. References: (1) S. Baghaee-Ravari et al. Eur. J. Plant Pathol. 129:413, 2001. (2) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (3) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St Paul, MN, 2001. (4) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.
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.
Understanding the mechanism of interaction between the oil palm and its key pathogen, Ganoderma spp. is crucial as the disease caused by this fungal pathogen leads to a major loss of revenue in leading palm oil producing countries in Southeast Asia. Here in this study, we assess the morphological and biochemical changes in Ganoderma disease infected oil palm seedling roots in both resistant and susceptible progenies. Rubber woodblocks fully colonized by G. boninense were applied as a source of inoculum to artificially infect the roots of resistant and susceptible oil palm progenies. Gas chromatography-mass spectrometry was used to measure an array of plant metabolites in 100 resistant and susceptible oil palm seedling roots treated with pathogenic Ganoderma boninense fungus. Statistical effects, univariate and multivariate analyses were used to identify key-Ganoderma disease associated metabolic agitations in both resistant and susceptible oil palm root tissues. Ganoderma disease related defense shifts were characterized based on (i) increased antifungal activity in crude extracts, (ii) increased lipid levels, beta- and gamma-sitosterol particularly in the resistant progeny, (iii) detection of heterocyclic aromatic organic compounds, benzo [h] quinoline, pyridine, pyrimidine (iv) elevation in antioxidants, alpha- and beta-tocopherol (iv) degraded cortical cell wall layers, possibly resulting from fungal hydrolytic enzyme activity needed for initial penetration. The present study suggested that plant metabolites mainly lipids and heterocyclic aromatic organic metabolites could be potentially involved in early oil palm defense mechanism against G. boninense infection, which may also highlight biomarkers for disease detection, treatment, development of resistant variety and monitoring.
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.