Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.
One hundred isolates of Phytophthora infestans collected from 10 provinces in China between 1998 and 2004 were analyzed for mating type, metalaxyl resistance, mitochondrial DNA (mtDNA) haplotype, allozyme genotype, and restriction fragment length polymorphism (RFLP) with the RG-57 probe. In addition, herbarium samples collected in China, Russia, Australia, and other Asian countries were also typed for mtDNA haplotype. The Ia haplotype was found during the first outbreaks of the disease in China (1938 and 1940), Japan (1901, 1930, and 1931), India (1913), Peninsular Malaysia (1950), Nepal (1954), The Philippines (1910), Australia (1917), Russia (1917), and Latvia (1935). In contrast, the Ib haplotype was found after 1950 in China on both potato and tomato (1952, 1954, 1956, and 1982) and in India (1968 and 1974). Another migration of a genotype found in Siberia called SIB-1 (Glucose-6-phosphate isomerase [Gpi] 100/100, Peptidase [Pep] 100/100, IIa mtDNA haplotype) was identified using RFLP fingerprints among 72% of the isolates and was widely distributed in the north and south of China and has also been reported in Japan. A new genotype named CN-11 (Gpi 100/111, Pep 100/100, IIb mtDNA haplotype), found only in the south of China, and two additional genotypes (Gpi 100/100, Pep 100/100, Ia mtDNA haplotype) named CN-9 and CN-10 were identified. There were more diverse genotypes among isolates from Yunnan province than elsewhere. The SIB-1 (IIa) genotype is identical to those from Siberia, suggesting later migration of this genotype from either Russia or Japan into China. The widespread predominance of SIB-1 suggests that this genotype has enhanced fitness compared with other genotypes found. Movement of the pathogen into China via infected seed from several sources most likely accounts for the distribution of pathogen genotypes observed. MtDNA haplotype evidence and RFLP data suggest multiple migrations of the pathogen into China after the initial introduction of the Ia haplotype in the 1930s.
Effects of phosphorus content (510 to 987 ppm) on the gelatinization and retrogradation of 6 potato cultivars (Benimaru, Hokkaikogane, Irish Cobbler, Konafubuki, Sakurafubuki, and Touya) were studied. Pasting properties were analyzed by RVA, thermal properties by DSC, and mechanical properties of the starch gels by TA. Phosphorus was positively correlated with swelling power (r= 0.84) and negatively correlated with solubility (r= 0.83). Phosphorus content showed significant effect on certain pasting properties of potato starch such as peak viscosity, breakdown, and setback. Phosphorus content showed a significant positive correlation with peak viscosity (r= 0.95) and breakdown (r= 0.90). Increasing concentration of phosphorus tends to decrease the setback. Phosphorus content had no influence on thermal properties and mechanical properties of potato starch gel.
Pectobacterium wasabiae was originally isolated from Japanese horseradish (Eutrema wasabi), but recently some Pectobacterium isolates collected from potato plants and tubers displaying blackleg and soft rot symptoms were also assigned to P. wasabiae. Here, combining genomic and phenotypical data, we re-evaluated their taxonomic position. PacBio and Illumina technologies were used to complete the genome sequences of P. wasabiae CFBP 3304T and RNS 08-42-1A. Multi-locus sequence analysis showed that the P. wasabiae strains RNS 08-42-1A, SCC3193, CFIA1002 and WPP163, which were collected from potato plant environment, constituted a separate clade from the original Japanese horseradish P. wasabiae. The taxonomic position of these strains was also supported by calculation of the in-silico DNA-DNA hybridization, genome average nucleotide indentity, alignment fraction and average nucleotide indentity values. In addition, they were phenotypically distinguished from P. wasabiae strains by producing acids from (+)-raffinose, α-d(+)-α-lactose, d(+)-galactose and (+)-melibiose but not from methyl α-d-glycopyranoside, (+)-maltose or malonic acid. The name Pectobacterium parmentieri sp. nov. is proposed for this taxon; the type strain is RNS 08-42-1AT (=CFBP 8475T=LMG 29774T).
l-Asparaginases have the potential to inhibit the formation of acrylamide, a harmful toxin formed during high temperature processing of food. A novel bacterium which produces l-asparaginase was screened. Type I l-asparaginase gene from Acinetobacter soli was cloned and expressed in Escherichia coli. The recombinant l-asparaginase had an activity of 42.0 IU mL-1 and showed no activity toward l-glutamine and d-asparagine. The recombinant l-asparaginase exhibited maximum catalytic activity at pH 8.0 and 40°C. The enzyme was stable in the pH ranging from 6.0 to 9.0. The activity of the recombinant enzyme was substantially enhanced by Ba2+, dithiothreitol, and β-mercaptoethanol. The Km and Vmax values of the l-asparaginase for the l-asparagine were 3.22 mmol L-1 and 1.55 IU μg-1, respectively. Moreover, the recombinant l-asparaginase had the ability to mitigate acrylamide formation in potato chips. Compared with the untreated group, the content of acrylamide in samples treated with the enzyme was effectively decreased by 55.9%. These results indicate that the novel type I l-asparaginase has the potential for application in the food processing industry.
Dickeya solani is an emerging pathogen that causes soft rot and blackleg diseases in several crops including Solanum tuberosum, but little is known about its genomic diversity and evolution.
BACKGROUND: Trichoderma sp. SL2 has been previously reported to enhance rice germination, vigour, growth and physiological characteristics. The use of Potato Dextrose Agar as carrier of Trichoderma sp. SL2 inoculant is not practical for field application due to its short shelf life and high cost. This study focuses on the use of corn and sugarcane bagasse as potential carriers for Trichoderma sp. SL2 inoculants.
FINDINGS: A completely randomized design was applied for this study. Trichoderma sp. SL2 suspension mixed with corn and sugarcane bagasse were used as treatment mixture in soil. Growth parameters including rice seedling height, root length, wet weight, leaf number and biomass were measured and compared to control. The results showed that Trichoderma sp. SL2 mixed with corn significantly enhanced rice seedlings root length, wet weight and biomass compared to Trichoderma sp. SL2 mixed with sugarcane bagasse and control.
CONCLUSION: Corn can be a potential carrier for Trichoderma spp. inoculants for field application.
BACKGROUND: To evaluate the impact of cold fermentation time on bagel rolls, the key aroma-active compounds in the volatile fractions obtained from three different bagel rolls through solvent assisted flavor evaporation (SAFE) were sequentially characterized by an aroma extract dilution analysis (AEDA), quantified by stable isotope dilution and analyzed by odor activity values (OAVs) respectively.
RESULTS: Findings revealed 40 aroma-active compounds with flavor dilution (FD) factor ranges of 2-1024. Of these, 22 compounds (FD ≥ 16) were quantified by stable isotope dilution assays (SIDA). Subsequent analysis of the 22 compounds by odor activity values (OAVs) revealed 14 compounds with OAVs ≥ 1 and the highest concentrations were obtained for 2,3-butanedione, 2-phenylethanol, 3-methylbutanal and acetoin respectively. Two recombination models of the bagels (i.e. 24 h and 48 h bagels) showed similarity to the corresponding bagels. Omission tests confirmed that 2,3-butanedione (buttery), acetoin (buttery), 2-acetyl-1-pyrroline (roasty), 5-methyl-2-furanmethanol (bread-like), (Z)-4-heptenal (biscuit-like) and 4-hydroxy-2,5-dimethyl-3(2H)-furanone, were the key aroma compounds. Additionally, acetic acid, butanoic acid, 2-phenylethanol (honey-like), 3-methylbutanoic acid, 2/3-methylbutanal, vanillin, 3-methylbutanol, methional were also important odorants of the bagel.
CONCLUSION: Whilst the long, cold fermented bagels exhibited roasty, malty, buttery, baked potato-like, smoky and biscuit-like notes, the control bagels produced similar but less intense odor notes.
Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. It has an optimal growth in yeast peptone dextrose (YPD) and yeast mould (YM) broth media but not in potato dextrose (PD) broth medium. Early phase G. antarctica PI12 cells had elongated-shape and became oval-shaped as they aged. G. antarctica PI12 exhibited bipolar budding and formed a chain of cells during the lag and early exponential phases. The number of chains decreased as the yeast aged. It appeared mainly as a single cell at the stationary phase, and a small number of them still produced buds. Some cells at the stationary phase entered the quiescence state (G0) as a longterm survival strategy. The G. antarctica PI12 cell size decreased when they entered the stationary phase. G. antarctica PI12 was found to produce hydrolytic enzymes, chitinase, cellulase, mannanase, and xylanase. A higher glucose concentration of 2% in the PD agar medium inhibited the activities of chitinase but not the cellulase, mananase and xylanase.
Cucumber (Cucumis sativus L.) is one of the most important vegetable fruits in Malaysia. Cucumber is principally grown in the states of Johor, Kelantan, and Perak. The broad host range Enterobacteriaceae pathogen, Pectobacterium carotovorum, can cause soft rot on stems or cucumber fruit. In Malaysia, cucumber is produced in a warm, humid climate, thus the plant is susceptible to attack by P. carotovorum at any time during production. In 2010, cucumber samples with wilted and chlorotic leaves, water-soaked lesions, and collapsed fruits were found in multiple fields. Small pieces of infected stems and fruit were immersed in 5 ml of saline solution (0.85% NaCl) for 20 min and then 50 μl of this suspension was spread onto nutrient agar (NA) and incubated at 27°C for 24 h. White-to-pale gray colonies with irregular margins were selected for analysis. For pathogenicity tests, cucumber fruits were surface sterilized by ethyl alcohol 70%, washed with sterilized distilled water, cut into small pieces, and inoculated with 20 μl of 108 CFU/ml suspensions of five representative strains. Cucumber plants were grown for 3 weeks in sterilized soil and their stems were inoculated with 20 μl of 108 CFU/ml of bacterial suspension. Inoculated samples and control (noninoculated) plants were placed in a growth chamber with 80 to 90% relative humidity at 27°C. Symptoms occurred on fruit slices and stems after 1 to 3 days and appeared the same as naturally infected samples, but the control samples remained healthy. Koch's postulates were fulfilled with the reisolation of cultures with the same characteristics as described earlier. Hypersensitivity reaction (HR) assays were done by infiltrating 108 CFU/ml of bacterial suspension into tobacco leaf epidermis and HR developed. All strains were subjected to biochemical and morphological assays, as well as molecular assessment. The strains were gram negative, facultative anaerobes, rod shaped, able to macerate potato slices and growth at 37°C; catalase positive; oxidase and phosphatase negative; able to degrade pectate; sensitive to erythromycin; negative for utilization of α-methyl glycoside, indole production, and reduction of sugars from sucrose; acid production from arabitol, sorbitol, and utilization of citrate were negative, but positive for raffinose and melibiose utilization. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment on agarose gel 1% (1). Amplification of intergenic transcribed spacer region by G1 and L1 primers gave two main bands at approximately 535 and 580 bp on agarose gel 1.5%. The ITS-PCR products were digested with RsaI restriction enzyme (3). On the basis of biochemical and morphological characteristics, PCR-based pel gene and characterization of the ITS region, and digestion of the ITS-PCR products with RsaI restriction enzyme, all isolates were identified as P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of soft rot caused by P. carotovorum subsp. carotovorum on cucumber 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 Press, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.
Passiflora edulis Sims f. edulis, known as purple passion fruit, is a woody, perennial vine that is grown for its attractive two-part flower and its purple, edible fruit (4). In November 2009, passion fruit vines were collected during a regulatory nursery inspection in Santa Barbara County and submitted to the California Department of Food and Agriculture Plant Pest Diagnostics Laboratory. Nearly 100% of the plants inspected, all of which were approximately 1.25 m tall, appeared stunted, defoliated, and severely wilted. Dark brown vascular discoloration was present in the roots and lower stems of the plants. A pinkish violet Fusarium oxysporum colony containing chlamydospores, multiseptate macroconidia, and microconidia formed on monophialidic conidiophores was consistently isolated from roots and stems onto half-strength acidified potato dextrose agar (aPDA). All further experiments were done with an isolate obtained from a single conidium. A portion of the translation elongation factor gene (TEF-1α) was amplified and sequenced with primers ef1 and ef2 from our isolate (GenBank No. JF332039) (3). BLAST analysis of the 615-bp amplicon with the FUSARIUM-ID database showed 99% similarity with a F. oxysporum passion fruit isolate from Australia (NRRL 38273) (3). To confirm pathogenicity, washed roots of four-leaf stage seedlings approximately 10 cm tall were submerged in a conidial spore suspension (106 spores/ml) for 15 min. The conidial suspension was prepared by flooding 10-day-old cultures grown on aPDA medium with sterile distilled water. Seven seedlings were inoculated and planted in 10-cm2 pots and kept in a 25°C growth chamber with a 12-h photoperiod. Seven seedlings were mock inoculated with sterile water. After 3 weeks, four of the seven inoculated plants had leaves with yellow veins and discolored roots and had partially defoliated. Two of the four symptomatic plants also had brown stem cankers. F. oxysporum grew from the isolated roots and stems of all the inoculated plants. F. oxysporum did not grow from root and stem pieces from the water-dipped plants and the plants remained asymptomatic. Inoculations were repeated on plants approximately 15 cm tall with F. oxysporum growing from roots and stem pieces of all inoculated plants. Symptoms of yellow veins and root necrosis were not observed until 4 weeks after inoculation. Fusarium wilt caused by F. oxysporum f. sp. passiflorae is a significant disease of P. edulis f. edulis in Australia. The disease has also been reported in South Africa, Malaysia, Brazil, Panama, and Venezuela; but it is unclear as to whether the symptoms were caused by Fusarium wilt or Haematonectria canker (1). Banana poka (P. mollissima), P. ligularis, and P. foetida are also susceptible hosts (2). To our knowledge, this is the first report of Fusarium wilt caused by F. oxysporum f. sp. passiflorae on passion fruit in North America. Passion fruit is not commercially produced for consumption in California so the economic importance of this disease appears to be limited to nursery production and ornamental landscapes. The grower of the California nursery stated that the infected passion fruit plants had been propagated on site from seed. The source of inoculum at this nursery remains unknown. References: (1) I. H. Fischer and J. A. M. Rezende. Pest Tech. 2:1, 2008 (2) D. E. Garder. Plant. Dis. 73:476, 1989. (3) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (4) F. W. Martin et al. Econ. Bot. 24:333, 1970.
Banana is the second largest cultivated fruit crop in Malaysia, and is cultivated for both the domestic market and also for export. Anthranose is a well-known postharvest disease of banana and with high potential for damaging market value, as infection commonly occurs during storage. Anthracnose symptoms were observed on several varieties of banana such as mas, berangan, awak, nangka, and rastali in the states of Perak and Penang between August and October 2011. Approximately 80% of the fruits became infected with initial symptoms characterized as brown to black spots that later became sunken lesions with orange or salmon-colored conidial masses. Infected tissues (5 × 5 mm) were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 3 to 5 min, rinsed with sterile distilled water, and plated onto potato dextrose agar (PDA). Direct isolation was done by transferring the conidia from conidial masses using an inoculation loop and plating onto PDA. For both methods, the PDA plates were incubated at 27 ± 1°C with cycles of 12 h light and 12 h darkness. Visible growth of mycelium was observed after 4 to 5 days of incubation. Twenty isolates with conidial masses were recovered after 7 days of incubation. The isolates produced grayish white to grayish green and grey to moss dark green colony on PDA, pale orange conidial masses, and fusiform to cylindrical and hyaline conidia with an average size of 15 to 19 × 5 to 6 μm. Appresoria were ovate to obovate, dark brown, and 9 to 15 × 7 to 12 μm and setae were present, slightly swollen at the base, with a tapered apex, and brown. The cultural and morphological characteristics of the isolates were similar to those described for C. gleosporioides (1,2,3). All the C. gloeosporioides isolates were deposited in culture collection at Plant Pathology Lab, University Sains Malaysia. For confirmation of the identity of the isolates, ITS regions were sequenced using ITS4 and ITS5 primers. The isolates were deposited in GenBank with accessions JX163228, JX163231, JX163201, JX163230, JX163215, JX163223, JX163219, JX163202, JX163225, JX163222, JX163206, JX163218, JX163208, JX163209, JX163210, JX431560, JX163212, JX163213, JX431540, and JX431562. The resulting sequences showed 99% to 100% similarity with multiple C. gloeosporioides isolates in GenBank. Pathogenicity tests were conducted using mas, berangan, awak, nangka, and rastali bananas. Fruit surfaces were sterilized with 70% ethanol and wounded using a sterile scalpel. Two inoculation techniques were performed separately: mycelia plug and conidial suspension. Mycelial disc (5 mm) and a drop of 20 μl spore suspension (106 conidia/ml) were prepared from 7-day-old culture and placed on the fruit surface. The inoculated fruits were incubated at 27 ± 1°C for 10 days at 96.1% humidity. After 3 to 4 days of inoculation, brown to black spotted lesions were observed and coalesced to become black sunken lesions. Similar anthracnose symptoms were observed on all banana varieties tested. C. gloeosporioides was reisolated from the anthracnose lesions of all the inoculated fruit in which the cultural and morphological characteristics were the same as the original isolates. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of Musa spp. in Malaysia. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) J. E. M. Mordue. Glomerella cingulata. CMI Description of Pathogenic Fungi and Bacteria, No. 315. CAB International,1971. (3) H. Prihastuti et al. Fungal Diversity 39:89, 2009.
Rambutan (Nephelium lappaceum L.) is among the tropical fruit grown in Malaysia and the demand for export rose in 2011. A fruit rot was observed between August and December 2011 from several areas in the states of Pulau Pinang and Perak, Malaysia. The symptoms initially appeared as light brown, water-soaked lesions that developed first in the pericarp and pulp, later enlarging and becoming dark brown. Greyish brown mycelia were observed on infected areas that turned yellowish at later stages of infection. Gliocephalotrichum bacillisporum was isolated from infected fruit by surface sterilization techniques. Conidia were mass-transferred onto potato dexstrose agar (PDA) plates and incubated at 27 ± 1°C. Tissue pieces (5 × 5 mm) excised from the margins between infected and healthy areas were then surface sterilized in 1% sodium hypochlorite for 3 to 5 min before being rinsed with distilled water, plated on PDA, and incubated at 27 ± 1°C for 7 days. Ten isolates of G. bacillisporum were obtained. Colonies on PDA were initially white before turning yellow with a feathery appearance. Microscopic characteristics on carnation leaf agar (CLA) consisted of hyaline conidia that were slightly ellipsoid to bacilliform with rounded apex ranging from 6.0 to 8.5 μm long and 2.0 to 2.5 μm wide. Conidiophores (70 to 130 μm long) were mostly single arising from large hypha approximately 13 to 16 μm. The conidiogenous structures were mostly quadriverticillate with dense, short, penicillate branches. The phialides were cylindrical and finger-like. Chlamydospores were present singly, in groups of 2 to 4, or in occasionally branched short chains and were brown in color with thick walls ranging from 11 to 13 μm. The cultural and morphological characteristics of G. bacillisporum isolates in the present study were very similar to previously published descriptions (1) except the conidiophores formed without sterile stipe extensions. All the G. bacillisporum isolates were deposited in culture collection at the Plant Pathology Lab, University Sains Malaysia, Penang. Molecular identification was accomplished from the ITS regions using ITS1 and ITS2 primers, and the β-tubulin gene using Bt2a and Bt2b primers (2). BLAST results from the ITS regions showed a 98 to 99% similarity with sequences of G. bacillisporum isolates reported in GenBank. Accession numbers of G. bacillisporum ITS regions: JX484850, JX484852, JX484853, JX484856, JX484858, JX484860, JX484862, JX484866, JX484867, and JX484868. The identity of G. bacillisporum isolates infecting rambutan was further confirmed by β-tubulin sequences (KC683909, KC683911, KC683912, KC683916, KC683919, KC683920, KC683923, KC683926, and KC683927), which showed 92 to 95% similarity with sequences of G. bacillisporum. Pathogenicity tests were also performed using mycelial plug (5 mm) and sprayed conidial suspensions (20 μl suspension of 106 conidia/ml) prepared from 7-day-old cultures. Inoculated fruits were incubated at 27 ± 1°C and after 10 days, similar rotting symptoms appeared on the fruit surface. The pathogen was reisolated from fruit rot lesions, thus fulfilling Koch's postulates, and tests were repeated twice. To our knowledge, this is the first report of G. bacillisporum causing fruit rot of rambutan (N. lappaceum L.) in Malaysia. References: (1) C. Decock et al. Mycologia 98:488, 2006. (2) N. L. Glass and G. C. Donaldson. Appl. Environ Microbiol. 61:1323, 1995.
Mangosteen (Garcinia mangostana L.) is a tropical evergreen tree that produces one of the most prized tropical fruits, commonly known as the "Queen of the Fruits.″ Mangosteen has the potential to occupy a rapidly expanding niche market in Hawaii. In October 2009, a disease was observed that produced brown leaf spots and blotches surrounded by bright yellow halos at a mangosteen orchard located in Hakalau, Hawaii (19° 53' 49″ N, 155° 7' 35″ W). Recently transplanted 10+ year old trees were 95 to 100% infected. Pieces of infected leaves and stems were surface-sterilized, plated on potato dextrose agar (PDA), and incubated at 24°C ± 1°C for 21 days. The fungus growing on PDA was pale buff with sparse aerial mycelium and acervuli containing black, slimy spore masses. Single spore isolates were used for the morphological characteristics and molecular analysis. Conidia were 5-celled. Apical and basal cells were hyaline; the three median cells were umber to olivaceous. Conidia (n = 50) were 24.3 ± 0.2 × 7.5 ± 0.1 μm, with apical appendages, typically three, averaging 24.3 ± 0.4 μm long, and a basal appendage averaging 6.7 ± 0.2 μm long. DNA sequences were obtained from the β-tubulin gene and the internal transcribed spacer (ITS1 and ITS2) and 5.8S regions of the rDNA to confirm the identification. The morphological descriptions and measurements were similar to P. virgatula (Kleb.) Steyaert (1). Although sequence data of the ITS region (GenBank Accession No. JN542546) supports the identity of the fungus as P. virgatula, the taxonomy of this genus remains confused since there are only a few type cultures, so it is impossible to use sequences in GenBank to reliably clarify species names (2). To confirm pathogenicity, six leaves of two 3-year-old seedlings were inoculated. Seven-day-old cultures grown on 10% V8 agar at 24°C under continuous fluorescent lighting were used for inoculations. The inoculum consisted of spore suspensions in sterile distilled water adjusted to 6 × 105 conidia/ml. Using a fine haired paint brush, the inoculum was brushed onto the youngest leaves, while sterile distilled water was used as the control. The plants were incubated in a clear plastic bag placed on the laboratory bench at 24°C for 48 hours, then placed on a greenhouse bench and observed weekly for symptoms. After 14 days, leaf spots ranging in size from pinpoint to 5.4 mm in diameter with a distinctive yellow halo were present. Within 35 days, the leaf spots enlarged to leaf blotches ranging in size from 11.5 × 13.3 mm up to 28.3 × 34.6 mm with brown centers and a distinctive yellow halo identical to the field symptoms. A Pestalotiopsis sp. identical to that used to inoculate the seedlings was recovered from the leaf spots and blotches, confirming Koch's postulates. The experiment was repeated twice. Pestalotiopsis leaf blight has been reported in other countries growing mangosteen, including Thailand, Malaysia, and North Queensland, Australia (3). However, to our knowledge, this is the first report of a Pestalotiopsis sp. causing a disease on mangosteen in Hawaii. Although this disease is considered a minor problem in the literature (3), effective management practices should be established to avoid potential production losses. References: (1) E. F. Guba. Monograph of Pestalotia and Monochaetia. Harvard University Press, Cambridge, MA. 1961. (2) S. S. N. Maharachchikumbura et al. Fungal Div. 50:167, 2011. (3) R. C. Ploetz. Diseases of Tropical Fruit Crops. CABI Publishing. Wallingford, Oxfordshire, UK, 2003.
In June 2011, lettuce (Lactuca sativa) plants cultivated in major lettuce growing areas in Malaysia, including the Pahang and Johor states, had extensive leaf spots. In severe cases, disease incidence was recorded more than 80%. Symptoms on 50 observed plants initially were as water soaked spots (1 to 2 mm in diameter) on leaves, and then became circular spots spreading over much of the leaves. In this research, main lettuce growing areas infected by the pathogen in the mentioned states were investigated and the pathogen was isolated onto potato dextrose agar (PDA). Colonies observed were greyish green to light brown. Single conidia were formed at the terminal end of conidiophores that were 28.8 to 40.8 μm long and 11.0 to 19.2 μm wide, and 2 to 7 transverse and 1 to 4 longitudinal septa. To produce conidia, the fungus was grown on potato carrot agar (PCA) and V8 juice agar media under 8-h/16-h light/dark photoperiod. Fourteen isolates were identified Stemphylium solani based on morphological criteria described by Kim et al. (1). To confirm morphological characterization, DNA of the fungus was extracted from mycelium and PCR was done using universal primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), which amplified the internal transcribed spacer (ITS) region of rDNA (2). The sequencing result was subjected to BLAST analysis which was 99% identical to the other published sequences in the GenBank database (GenBank Accession Nos. AF203451 and HQ840713). The nucleotide sequence was deposited in GenBank under Accession No. JQ736022. Pathogenicity testing of representative isolate was done using 20 μl of conidial suspension with a concentration of 1 × 105/ml in droplets (three drops on each leaf) on four detached 45-day-old lettuce leaves cv. BBS012 (3). Fully expended leaves were placed on moist filter paper in petri dishes and were incubated in humid chambers at 25°C. The leaves inoculated with sterile water served as control. After 7 days, disease symptoms were observed, which were similar to those symptoms collected in infected fields and the fungus was reisolated and confirmed as S. solani based on morphological criteria (1) and molecular characterization (2). Control leaves remained healthy. Pathogenicity testing was completed twice. To our knowledge, this is the first report of S. solani on lettuce in Malaysia and it may become a serious problem because of its broad host range, variability in pathogenic isolates, and prolonged active phase of the disease cycle. Previous research has shown that S. solani is a causal agent of gray leaf spot on lettuce in China (4). References: (1) B. S. Kim et al. Plant Pathol. J. 20:85, 2004. (2) Y. R. Mehta et al. Current Microbiol. 44:323, 2002. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) F. L. Tai. Sylloge Fungorum Sinicorum, Sci. Press, Acad. Sin., Peking, 1979.
Bok choy (Brassica chinensis L.) is a temperate vegetable grown in the cool highland areas of Malaysia. In June 2010, vegetable growing areas of the Cameron Highlands, located in Pahang State, Malaysia, were surveyed for the prevalence of anthracnose disease caused by Colletotrichum species. Diseased samples were randomly collected from 12 infested fields. Anthracnose incidence on bok choy varied from 8 to 36% in different nursery fields. Disease symptoms initially appeared as small water-soaked spots scattered on the leaf petioles of young plants. As these spots increased in size, they developed irregular round spots that turned to sunken grayish brown lesions surrounded by brownish borders. When the lesions were numerous, leaves collapsed. Pale buff to salmon conidial mass and acervuli were observed on well-developed lesions. The acervuli diameter varied in size from 198 to 486 μm, averaging 278.5 μm. Morphological and cultural characteristics of the fungus were examined on potato dextrose agar incubated for 7 days at 25 ± 2°C under constant fluorescent light. Vegetative mycelia were hyaline, septate, branched, and 2 to 7 μm in diameter. The color of the fungal colonies was grayish brown. Conidia were hyaline, aseptate, falcate, apices acute, and 21.8 to 28.5 × 2.6 to 3.4 mm. Setae were pale brown to dark brown, 75 to 155 μm long, base cylindrical, and tapering towards the acute tip. Appressoria were solitary or in dense groups, light to dark brown, entire edge to lobed, roundish to clavate, 6.5 to 14 × 5.8 to 8.6 μm, averaging 9.2 × 6.8 μm, and had a L/W ratio of 1.35. Based on the keys outlined by Mordue 1971 (2) and Sutton 1980 (3), the characteristics of this fungus corresponded to Colletotrichum capsici. Sequence analysis of the ITS-rDNA obtained from the Malaysian strain CCM3 (GenBank Accession No. JQ685746) using primers ITS5 and ITS4 (1) when aligned with deposited sequences from GenBank revealed 99 to 100% sequence identity with C. capsici strains (DQ286158, JQ685754, DQ286156, GQ936210, and GQ369594). A representative strain CCM3 was used for pathogenicity testing. Four non-infected detached leaves of 2-week-old B. chinensis were surface-sterilized and inoculated by placing 10 μl of conidial suspension (106 conidia ml-1) using either the wound/drop or non-wound/drop method, and distilled water was used as a control (1). Leaves were incubated at 25°C, 98% RH. The experiment was repeated twice. Five days after inoculation, typical anthracnose symptoms with acervuli formation appeared on the surface of tissues inoculated with the spore suspension, but not on the water controls. A fungus with the characteristics of C. capsici was recovered from the lesions on the inoculated leaves. Anthracnose caused by C. capsici has been reported on different vegetable crops, but not on bok choy (3). To the best of our knowledge, this is the first report of C. capsici causing anthracnose on bok choy in Malaysia. References: (1) R. Ford et al. Aust. Plant Pathol. 33:559, 2004. (2) J. E. M. Mordue. CMI Description of Pathogenic Fungi and Bacteria. Commonwealth Mycol. Inst., Kew, UK. 1971. (3) B. C. Sutton. The Genus Glomerella and its anamorph Colletotrichum. CAB International, Wallingford, UK, 1992. (4) P. P. Than et al. Plant Pathol. 57:562, 2008.
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.
In January 2011, branch samples were collected from langsat (Lansium domesticum Corr.), a fruit from Southeast Asia with an expanding niche market in Hawaii, exhibiting corky bark symptoms similar to that found on rambutan (Nephelium lappaceum) and litchi (Litchi chinensis) (3). The orchard, located along the Hamakua Coast of Hawaii Island, had 5- to 10-year-old trees, all with corky bark symptoms. As the trees matured, the cankers increased in size and covered the branches and racemes, often resulting in little to no fruit production. Scattered along the infected bark tissue were elongated, black ascomata present in the cracks. Ascomata were removed from the cracks using a scalpel blade, placed at the edge of a water agar petri dish and gently rolled along the agar surface to remove bark tissue and other debris. Individual ascomata were placed in 10-μl drops of 10% sodium hypochlorite on fresh water agar for 20 s, removed, and placed on potato dextrose agar petri dishes amended with 25 μg/ml streptomycin. The isolates were kept at 24°C under continuous fluorescent lighting. After 9 days, black pycnidia were present, which produced smooth, hyaline, linear to curved, filiform conidia, 4 to 6 septate (mostly 6), 31.8 to 70.1 × 2.0 to 2.8 μm. The morphological descriptions and measurements were similar to those reported for Dolabra nepheliae (3). The nucleotide sequence of the internal transcribed spacer (ITS) region including ITS1, 5.8S, and ITS2 intergenic spacers was determined for strain P11-1-1and a BLAST analysis of the sequence (GenBank Accession No. JX566449) revealed 99% similarity (586/587 bp) with the sequence of D. nepheliae strain BPI 882442 on N. lappaceum from Honduras. Based on morphology and ITS sequencing, the fungus associated with the cankers was identified as the same causal agent reported on rambutan and pulasan (N. mutabile) from Malaysia (1), and later reported on rambutan and litchi in Hawaii and Puerto Rico (3). Upon closer observations of the diseased samples, sections of corky bark contained at least two larval insects. The beetles were identified as Corticeus sp. (Coleoptera: Tenebrionidae) and Araecerus sp. (Coleoptera: Anthribidae) by the USDA-ARS Systematic Entomology Laboratory (Beltsville, MD). A corky bark disease on the trunk and larger limbs of mature langsat trees in Florida was thought to be caused by Cephalosporium sp. with larvae (Lepidoptera: Tineidae) feeding on the diseased tissue (2). It is not known the extent to which either of the beetle species is associated with L. domesticum in Hawaii or if they play a role in the bark disorder. To our knowledge, this is the first report of Dolabra nepheliae being found on langsat in Hawaii. Effective management practices should be established to avoid potential production losses or spreading the disease to alternative hosts. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) J. Morton. Langsat. In: Fruits of Warm Climates, p. 201-203. Julia F. Morton, Miami, FL, 1987. (3) A. Y. Rossman et al. Plant Dis. 91:1685, 2007.
Soft rot of cabbage (Brassica rapa) occurs sporadically in Malaysia, causing economic damage under the hot and wet Malaysian weather conditions that are suitable for disease development. In June 2011, 27 soft rotting bacteria were isolated from cabbage plants growing in the Cameron Highlands and Johor State in Malaysia where the economic losses exceeded 50% in severely infected fields and greenhouses. Five independent strains were initially identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and elicit hypersensitive reaction (HR) on Nicotiana tabaccum and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG-positive and positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. All strains were negative for indole production, phosphatase activity, reducing sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-methyl-D-glucoside, and D-arabitol. All the strains exhibited pectolytic activity on potato slices. PCR assays were conducted to distinguish P. wasabiae from P. carotovorum subsp. brasiliensis, P. atrosepticum, and other Pectobacterium species using primers Br1f/L1r (2), Eca1f/Eca2r (1), and EXPCCF/EXPCCR, respectively. DNA from strains did not yield the expected amplicon with the Br1f/L1r and Eca1f/Eca2r, whereas a 550-bp amplicon typical of DNA from P. wasabiae was produced with primers EXPCCF/EXPCCR. ITS-RFLP using the restriction enzyme, Rsa I, produced similar patterns for the Malaysian strains and the P. wasabiae type strain (SCRI488), but differentiated it from P. carotovora subsp. carotovora, P. atrosepticum, P. carotovorum subsp. brasiliensis, and Dickeya chrysanthemi type strains. BLAST analysis of the 16S rRNA DNA sequence (GenBank Accession No. KC445633) showed 99% identity to the 16S rRNA of Pw WPP163. Phylogenetic reconstruction using concatenated DNA sequences of mdh and gapA from P. wasabiae Cc6 (KC484657) and other related taxa (4) clustered Malaysian P. wasabiae strains with P. wasabiae SCRI488, readily distinguishing it from other closely related species of Pectobacterium. Pathogenicity assays were conducted on leaves and stems of four mature cabbage plants for each strain (var. oleifera) by injecting 10 μl of a bacterial suspension (108 CFU/ml) into either stems or leaves, and incubating them in a moist chamber at 80 to 90% relative humidity at 30°C. Water-soaked lesions similar to those observed in the fields and greenhouses were observed 72 h after injection and bacteria with similar characteristics were consistently reisolated. Symptoms were not observed on water-inoculated controls. The pathogenicity test was repeated with similar results. P. wasabiae was previously reported to cause soft rot of horseradish in Japan (3). However, to our knowledge, this is the first report of P. wasabiae infecting cabbage in Malaysia. References: (1) S. H. De Boer and L. J. Ward. Phytopathology 85:854, 1995. (2) V. Duarte et al. J. Appl. Microbiol. 96:535, 2004. (3) M. Goto and K. Matsumoto. Int. J. Syst. Bacteriol. 37:130, 1987. (4) B. Ma et al. Phytopathology 97:1150, 2007.
In 2011, a severe gray leaf spot was observed on eggplant (Solanum melongena) in major eggplant growing areas in Malaysia, including the Pahang, Johor, and Selangor states. Disease incidence was >70% in severely infected areas of about 150 ha of eggplant greenhouses and fields examined. Symptoms initially appeared as small (1 to 5 mm diameter), brownish-black specks with concentric circles on the lower leaves. The specks then coalesced and developed into greyish-brown, necrotic lesions, which also appeared on the upper leaves. Eventually, the leaves senesced and were shed. Tissue cut from the edges of leaf spots were surface-sterilized in 1% NaOCl for 2 min, rinsed in sterilized water, dried, and incubated on potato dextrose agar (PDA). Fungal colonies were greyish green to light brown, and produced a yellow pigment. Single, muriform, brown, oblong conidia formed at the terminal end of each conidiophore, were each 21.6 to 45.6 μm long and 11.5 to 21.6 μm wide, and contained 2 to 7 transverse and 1 to 4 longitudinal septa. The conidiophores were tan to light brown and ≤220 μm long. Based on these morphological criteria, 25 isolates of the fungus were identified as Stemphylium solani (1). To produce conidia in culture, 7-day-old single-conidial cultures were established on potato carrot agar (PCA) and V8 juice agar media under an 8-h/16-h light/dark photoperiod at 25°C (4). Further confirmation of the identification was obtained by molecular characterization in which fungal DNA was extracted and the internal transcribed spacer (ITS) region of ribosomal DNA amplified using primers ITS5 and ITS4 (2), followed by direct sequencing. A BLAST search in the NCBI database revealed that the sequence was 99% identical with published ITS sequences for two isolates of S. solani (Accession Nos. AF203451 and HQ840713). The amplified ITS region was deposited in GenBank (JQ736023). Pathogenicity testing of a representative isolate was performed on detached, 45-day-old eggplant leaves of the cv. 125066-X under laboratory conditions. Four fully expanded leaves (one wounded and two non-wounded leaflets/leaf) were placed on moist filter paper in petri dishes, and each leaflet inoculated with a 20-μl drop of a conidial suspension containing 1 × 105 conidia/ml in sterilized, distilled water (3). The leaves were wounded by applying pressure to leaf blades with the serrated edge of forceps. Four control leaves were inoculated similarly with sterilized, distilled water. Inoculated leaves were incubated in humid chambers at 25°C with 95% RH and a 12-h photoperiod. After 7 days, symptoms similar to those observed in the original fields developed on both wounded and non-wounded inoculated leaves, but not on control leaves, and S. solani was reisolated consistently from the symptoms using the same method as the original isolations. Control leaves remained asymptomatic and the fungus was not isolated from these leaves. The pathogenicity testing was repeated with similar results. To our knowledge, this is the first report of S. solani on eggplant in Malaysia. References: (1) B. S. Kim et al. Plant Pathol. J. 20:85, 2004. (2) Y. R. Mehta et al. Curr. Microbiol. 44:323, 2002. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) E. G. Simmons. CBS Biodiv. Series 6:775, 2007.