Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding N-acetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.

An understanding of anthracnose (Colletotrichum acutatum) infections, including the infection of flowers and latent infection early in the season, is necessary to achieve successful control by means of properly timed spraying with a curative fungicide. In the present study, latent anthracnose infection of chili was investigated under greenhouse and field conditions in 2007-2008. Flowers on greenhouse-grown seedlings were infected and 11% of the young fruits subsequently showed symptoms of anthracnose. Apparently healthy-looking green peppers obtained from unsprayed fields or an organic market also exhibited symptoms of anthracnose after 4 days of incubation under high moisture conditions at $25^{\circ}C$; less than 1% of the peppers were found to be latently infected. To determine the natural timing of infection in the field, 3,200 fruits were wrapped in paper bags and then selectively unwrapped and examined for signs of infection. Field experiments were conducted at Suwon (cvs. Yokkang, Manitta, Olympic) and Asan (cv. Chunhasangsa) in 2008. The 7- to 10-day wrapping periods were July 25-31, July 31-August 7, August 7-15, August 15-24, and August 24-September 3. The 1-to 2-month wrapping periods were from July 4, July 31, and August 15 until harvest (Sept. 3). The controls consisted of 1,712 field-grown non-wrapped fruits. The rates of infection on the various cultivars were Yokkang 55%, Manitta 37%, Olympic 55%, and Chunhasangsa 20%. A distinct period in which anthracnose infection suddenly increased could not be identified; however, attempts to guess the approximate timing of field infection showed that 0-39% of the plants had latent infections, while depending on the cultivar, 8-14% of the plants examined in August and 4-13.5% of the those examined during May-July showed symptoms of infection. Delaying fungicide spraying by 24 and 48 h after artificial infection decreased the rates of infection by 10% and 25-30%, respectively. Chemical control of anthracnose based on a forecasting model should be considered starting from the transplanting stage, with spraying within a day after warning and care being taken not to latently infect apparently healthy pepper fruits.

The causal agents of bacterial blossom blight in kiwifruit were isolated from flowers displaying symptoms in Korea. The pathogens were characterized by biochemical and physiological tests, and identified on the basis of 16S rDNA and 16S-23S internal transcribed spacer (ITS) sequences. Pathogenicity tests demonstrated that the blossom blight of kiwifruit in Korea is caused by two pathogens, Pseudomonas syringae pv. syringae and P. fluorescens. Carbon source utilization and DNA-DNA hybridization experiments confirmed P. fluorescens as one of the causal agents of blossom blight of kiwifruit. P. syringae pv. syringae and P. fluorescens can be distinguished from each other by the symptoms they produce in flowers. P. syringae pv. syringae primarily affected the stamen, while P. fluorescens caused rotting of all internal tissues of buds or flowers.

Lily mottle virus (LMoV) causes flower quality reduction in Lilium spp. The coat protein gene was RT-PCR-amplified from total RNA extracted from infected lily leaves and the amplified fragment was cloned into the pRSET expression vector tagged with a His-MBP. The plasmid of recombinant coat protein was used to transform an Escherichia coli strain pLysS and was expressed. The coat protein was purified by affinity chromatography using a Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE. The in vitro-expressed protein was used for immunization of mice. The polyclonal and monoclonal antibodies reacted specifically for the detection of LMoV in lily extracts in Western blot. Moreover the monoclonal antibodies reacted with lily extracts in DAS-ELISA with no unspecific or heterologous reactions against other non-serologically related viruses, but the polyclonal antibodies revealed a weak reaction against both infected lily and healthy control.

Viral infections and root growth were examined to elucidate the relationship between viral resistance and root growth in 26 Korean hulless barley cultivars. Viral resistance was estimated in experimental filed of Honam agricultural research institute for 3 years. Length and number of seminal and adventitious roots were examined for evaluation of root growth in both field and green-house conditions 30 days after seeding. Dominant viral infection occurred in Korean hulless barley by Barley yellow mosaic virus (BaYMV) in fields; however, susceptible cultivars were infected by either BaYMV, Barley mild mosaic virus (BaMMV) or both. Only four cultivars, including Donghanchalssalbori, Kwangwhalssalbori, Namhossalbori and Naehanssalbori, presented stable resistance to viral infections. Susceptible cultivars to viral infection in fields showed shorter seminal root length and fewer adventitious root number than resistant cultivars. Resistant cultivars showed better root growth and significant difference in adventitious root length in green house conditions. Increase in the number of seminal roots in resistant cultivars was derived from decreased damage of roots by the viral infection compared to the susceptible cultivars.

Tylenchulus semipenetrans, citrus nematode is an important phytopathogenic nematode and responsible for serious damage on citrus. However, little information is available about genetic variability of T. semipenetrans among different populations with variation of conventional diagnostic characteristics. In this study, we compared the morphometric and genetic characteristics among different populations. The mature female of T. semipenetrans collected in this study had thicker cuticle than those in the previous studies. In comparative sequence analysis of T. semipenetrans populations obtained from Jeju in Korea, we observed genetic variations within clones generated from single individuals. To determine whether variability among copies of nuclear ribosomal DNA sequences exists in the genome of T. semipenetrans, PCR-RFLP technique from individuals of Korean isolates with MseI and MspI restriction enzymes was used to prove experimentally that all populations have intra-specific variations. Restriction enzyme digestion created several fragments on 3.0% agarose gel corresponding to several haplotypes in all populations, though some populations displayed fragment deletion. The total length of fragments was larger than before digestion, indicating sequence heterogeneity within the genome of T. semipenetrans.

Korean pine (Pinus koraiensis Sieb. et Zucc.), a five-needle pine, has recently been suffering pine wilt disease caused by non-native pinewood nematode, Bursaphelenchus xylophilus. Three-year-old Korean pine seedlings were inoculated with 10,000 pathogenic nematodes in a greenhouse to investigate disease development, water content and the density of nematodes in stems. Needle dehydration, xylem drying and pith browning started 20 days after inoculation (DAI). There were significant differences between seedlings inoculated with nematodes and control seedlings in the relative water content of stems and leaves at 20 and 30 DAI. At 60 DAI, all remaining seedlings inoculated with nematodes had died, but control seedlings all remained alive. The average number of nematodes recovered from stems of Korean pine dramatically increased from 10 to 20 DAI, and then decreased at the end of the experiment at 60 days. This study suggests that the relative water content of stems and leaves in current-year branches could be used as a useful physiological indicator for early diagnosis of pine wilt disease.

Root-knot nematode species, such as Meloidogyne hapla, M. incognita, M. arenaria, and M. javanica are the most economically notorious nematode pests, causing serious damage to a variety of crops throughout the world. In this study, DNA sequence analyses were performed on the D3 expansion segment of the 28S gene in the ribosomal DNA in an effort to characterize genetic variations in the three Meloidogyne species obtained from Korea and four species from the United States. Further, PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), SCAR (Sequence Characterized Amplified Region) PCR and RAPD (Randomly Amplified Polymorphic DNA) were also utilized to develop methods for the accurate and rapid species identification of the root-knot nematode species. In the sequence analysis of the D3 expansion segment, only a few nucleotide sequence variations were detected among M. incognita, M. arenaria, and M, javanica, but not M. hapla. As a result of our haplotype analysis, haplotype 5 was shown to be common in M. arenaria, M. incognita, M. javanica, but not in the facultatively parthenogenetic species, M. hapla. PCR-RFLP analysis involving the amplification of the mitochondrial COII and large ribosomal RNA (lrRNA) regions yielded one distinct amplicon for M. hapla at 500 bp, thereby enabling us to distinguish M. hapla from M. incognita, M. arenaria, and M. javanica reproduced via obligate mitotic parthenogenesis. SCAR markers were used to successfully identify the four tested root-knot nematode species. Furthermore, newly attempted RAPD primers for some available root-knot nematodes also provided some species-specific amplification patterns that could also be used to distinguish among root-knot nematode species for quarantine purposes.

The potential of. nonpathogenic Fusarium oxysporum strain Avr5, either alone or in combination with chitosan and Bion, for inducing defense reaction in tomato plants inoculated with F. oxysporum f. sp lycopersici, was studied in vitro and glasshouse conditions. Application Bion at concentration of 5, 50, 100 and $500{\mu}g$/ml, and the highest concentration of chitosan reduced in vitro growth of the pathogen. Nonpathogenic F. oxysporum Avr5 reduced the disease severity of Fusarium wilt of tomato in split plants, significantly. Bion and chitosan applied on tomato seedlings at concentration $100{\mu}g$ a.i./plant; 15, 10 and 5 days before inoculation of pathogen. All treatments significantly reduced disease severity of Fusarium wilt of tomato relative to the infected control. The biggest disease reduction and increasing tomato growth belong to combination of nonpathogenic Fusarium and Bion. Growth rate of shoot and root markedly inhibited in tomato plants in response to tomato Fusarium wilt as compared with healthy control. These results suggest that reduction in disease incidence and promotion in growth parameters in tomato plants inoculated with nonpathogenic Fusarium and sprayed with elicitors could be related to the synergistic and cooperative effect between them, which lead to the induction and regulation of disease resistance. Combination of elicitors and non-pathogenic Fusarium synergistically inhibit the growth of pathogen and provide the first experimental support to the hypothesis that such synergy can contribute to enhanced fungal resistance in tomato. This chemical could provide a new approach for suppression of tomato Fusarium wilt, but its practical use needs further investigation.

Bacterial wilt, Fusarium wilt and Foot rot caused by Ralstonia solanacearum, Fusarium oxysporum, and Phytophthora capsici respectively, continue to be severe problems to tomato, potato and black pepper growers in Vietnam. Three bio-products, Bacillus vallismortis EXTN-1 (EXTN-1), Bacillus sp. and Paenibacillus sp. (ESSC) and Bacillus substilis (MFMF) were examined in greenhouse bioassay for the ability to reduce bacterial wilt, fusarium wilt and foot rot disease severity. While these bio-products significantly reduced disease severities, EXTN-1 was the most effective, providing a mean level of disease reduction 80.0 to 90.0% against bacterial wilt, fusarium wilt and foot rot diseases under greenhouse conditions. ESSC and MFMF also significantly reduced fusarium wilt, bacterial wilt and foot rot severity under greenhouse conditions. Bio-product, EXTN-1 with the greatest efficacy under greenhouse condition was tested for the ability to reduce bacterial wilt, fusarium wilt and foot rot under field condition at Song Phuong and Thuong Tin locations in Ha Tay province, Vietnam. Under field condition, EXTN-1 provided a mean level of disease reduction more than 45.0% against all three diseases compared to water treated control. Besides, EXTN-1 treatment increased the yield in tomato fruits 17.3% than water treated control plants.

During the course of a study to develop a spraying program at 15-day spray intervals, two important findings were identified allowing for further reduction of spray frequency by increasing the spray interval. In evaluating the contribution of fungicides from a 15-day spray interval program, control of white rot, which is of prime importance in Korea, was not affected, in spite of the extended spray interval caused by omitting the fungicides during the season. In another experiment assessing the duration of the protective activities of several key fungicides used in the 15-day spray interval program, infection control was maintained for almost 30 days for some fungicide. Based on these two findings, a basic spraying program with a 25-day spray interval was developed. This program was modified for four successive years to improve the control efficacy against bitter rot and Marssonina blotch, which sometimes causes as much damage as white rot.

Powdery and downy mildews caused by Sphaerotheca fusca and Pseudoperonospora cubensis are the most common and serious diseases of cucumber worldwide. In spite of the introduction of highly effective systemic fungicides, control of these diseases remains elusive. Hence, this study aimed to develop an alternative method to chemicals in controlling the diseases by using different types of cooking oil. Egg yolk, which contains a natural emulsifier, lecithin, was selected as a surfactant to emulsify the oils. Among the different cooking oils used, soybean, canola (rape seed), safflower, sunflower, olive, and corn oils showed over 95% control values against powdery mildew of cucumber in a greenhouse test. In particular, 0.3% canola oil emulsified with 0.08% yolk (1 yolk and 60 ml canola in 20 l spray) was found to be the most effective. The treatment resulted in 98.9% and 96.3% control efficacies on powdery and downy mildews, respectively, of cucumber in the field. Canola oil exhibited direct and systemic effect, wherein powdery mildew of cucumber was suppressed only on treated leaves but not on non-treated leaves in a plant, while mycelia and conidia of the pathogen were severely distorted or destroyed by the treatment. The prospect of using the canola oil and yolk mixture as a natural fungicide is highly promising because of its effectiveness, availability, low cost, simple preparation, and safety to humans and the environment. The use of the canola oil and yolk mixture is expected to be an effective fungicide for use in organic farming and home gardening.

The stability of three different kinds of pUC-derived plasmids, pDsRed, pZsYellow, and pGFPuv, was investigated in Pectobacterium strains to utilize those plasmids as tracers. All three plasmids pDsRed, pZsYellow and pGFPuv showed their specific colors in Pectobacterium strains. Especially, the plasmid pDsRed conferred bright pink colonies on the Pectobacterium strains. When the bacteria lost the plasmid pDsRed, the colonies turned white, suggesting that the plasmid could be a good marker system for Pectobacterium strains on different environmental conditions. The effect of the antibiotic pressure on the stability of the plasmid was different depending on the host bacteria. P. carotovorum subsp. betavasculorum was more sensitive to the antibiotic pressure than P. carotovorum subsp. carotovorum Pcc21. However, temperature change significantly affected plasmid stability on both Pectobacterium strains. Almost all strains lost the plasmids with the shift in temperature from $28^{\circ}C$ to $37^{\circ}C$. Presence of the plasmids did not affect bacterial pathogenicity on their own host plants. Among three plasmids, pZsYellow was not useful as a marker because the yellow fluorescent proteins from pZs Yellow were interfered with the yellow natural fluorescence of the plant tissues induced by the defense system. Since the red color of DsRed can be seen with naked eyes, plasmid pDsRed was applicable as a marker. However, the color change was slow so that additional manipulation to increase the expression speed was necessary. Plasmid pGFPuv could serve as a perfect marker without any problem, tracing the reproduction and spread of the plant pathogens perfectly.

The low virus titer in woody plant tissues and the presence of inhibitor compounds such as polyphenols, tannins and polysaccharides are common difficulties that compromise purification of plant viroids from their woody hosts. A simple, reliable method of RNA isolation using CF11 cellulose column on a microcentrifuge tube scale for detecting Apple scar skin viroid (ASSVd) in apple was developed. Total RNA extracted from leaf, woody bark and the fruit skin was used for reverse transcription. RT-PCR products could be detected from RNA prepared from dormant woody bark, fruit skin and fresh leaves with both the CF11 cellulose column method and NucliSens extractor in February, August and November. Meanwhile, with the RNeasy kit RT-PCR, products were detected only in leaves and not from bark or fruit skin. The PCR product, about 330 base pairs, was analyzed by agarose gel electrophoresis. The CF11 cellulose column method was effective for detecting ASSVd. The method enabled the processing of a large numbers of samples of dormant woody bark, leaf and fruit skin of apple.

Stem canker symptoms were observed in a rape field in Muan, Korea during a disease survey in May 2006. A total of 15 isolates of Phoma sp. were obtained from the infected stems of the plant. All isolates were identified as Leptosphaeria biglobosa based on their morphological and cultural characteristics. The Korean isolates of L. biglobosa were assigned to 'brassicae' among six subclades of L. biglobosa complex based on the entire ITS sequences of rDNA. Pathogenicity of the fungal isolates was confirmed on leaves and stems of rape by artificial inoculation. This is the first report that Leptosphaeria biglobosa causes stem canker of rape in Korea.

Powdery mildew symptoms were frequently on plants of goat's-beard (Aruncus dioicus var. kamtschaticus) found cultivated in the island Ulleungdo in June 2008. The symptoms appeared as powdery mycelial colonies and dark brown cleistothecia on leaves, petioles, stems and budding flowers of the plant. Incidence of the disease was as high as 50-80% in the goat's-beard fields. Specimens of the diseased plants were collected and examined for morphological characteristics of the causal fungus. On the basis of morphological characteristics of the conidial stage and the teleomorph, the fungus was identified as Podosphaera ferruginea. This is the first report of goat's-beard powdery mildew caused by Podosphaera ferruginea in Korea.

See Full Text