Identification and Characterization of Cercospora malayensis Causing Leaf Spot on Kenaf.

In September 2013 and 2014, a significant number of kenaf plants showing symptoms of leaf spots with approximately 50% incidence were found in experimental plots in Iksan and Namwon, Korea. Leaf spots were circular to irregular, more or less vein-limited, reaching to 10 mm in diameter. The spots were initially uniformly brown to reddish brown, turning pale brown with a purplish margin and showing grayish patches on the lesion due to heavy fructification. The causative agent of the leaf spot disease was identified as Cercospora malayensis. The pathogenicity test was conducted with similar results, which fulfilled Koch's postulates. This is the first report of C. malayensis infection of kenaf in Korea.

The Development of Simple Methods for the Maintenance and Quantification of Polymyxa graminis.

Polymyxa graminis, a root endoparasite of several cereal species, is considered to be non-pathogenic but serves as a vector of various plant viruses belonging to the genera Bymovirus, Furovirus, and Pecluvirus. Specifically, it reduces barley productivity by transmitting the Barley Yellow Mosaic Virus (BaYMV). To date, due to its obligate biotrophic property, no artificial culturing of P. graminis was reported and its quantification was also technically challenging. Here, we developed a novel and simple method to infect P. graminis within sterile barley roots in contamination free by preparing nearly pure zoospore inoculum. Such artificial maintenance of P. graminis was verified based on the presence of various developmental stages in infected barley roots under microscope. In addition, the population of resting spores in host tissue was determined by establishing standard curve between manually counted number of spores and Ct values of 18S rDNA amplification using quantitative real-time PCR. Furthermore, it was validated that standard curve generated was also applicable to estimate the abundance of P. graminis in soil environments. In conclusion, the present study would help to generate a system to investigate the etiological causes as well as management of plant diseases caused by P. graminis and BaYMV in tissue and soil.

Characterization of a Septobasidium sp. Associated with Felt Disease of Schisandra chinensis.

Extensive disease surveys performed during the summers of 2013 and 2014 in Schisandra chinensis orchards resulted in the finding of a Septobasidium sp. associated with felt disease. The fungus was characterized to be symbiotic with a scale insect (Pseudaulacaspis cockerelli). Morphological and molecular characteristics of the Septobasidium isolates were investigated. The isolates were morphologically and phylogenetically close to S. bogoriense. We tentatively describe this isolate as a Septobasidium sp., mainly because of the limited amount of information available on the internal transcribed spacer region of the ribosomal DNA of Septobasidium spp.

Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification.

The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

First Report on Fusarium Wilt of Zucchini Caused by Fusarium oxysporum, in Korea.

Fusarium wilt of zucchini in Jeonju, Korea, was first noticed in May 2013. Symptoms included wilting of the foliage, drying and withering of older leaves, and stunting of plants. Infected plants eventually died during growth. Based on morphological characteristics and phylogenetic analyses of the molecular markers (internal transcribed spacer rDNA and translation elongation factor 1α), the fungus was identified as Fusarium oxysporum. Pathogenicity of a representative isolate was demonstrated via artificial inoculation, and it satisfied Koch's postulates. To our knowledge, this is the first report of F. oxysporum causing wilt of zucchini in Korea.

Identification and Characterization of the Causal Organism of Gummy Stem Blight in the Muskmelon (Cucumis melo L.).

Gummy stem blight is a major foliar disease of muskmelon (Cucumis melo L.). In this study, morphological characteristics and rDNA internal transcribed spacer (ITS) sequences were analyzed to identify the causal organism of this disease. Morphological examination of the Jeonbuk isolate revealed that the percentage of monoseptal conidia ranged from 0% to 10%, and the average length × width of the conidia was 70 (± 0.96) × 32.0 (± 0.15) µm on potato dextrose agar. The BLAST analysis showed nucleotide gaps of 1/494, 2/492, and 1/478 with identities of 485/492 (98%), 492/494 (99%), 491/494 (99%), and 476/478 (99%). The similarity in sequence identity between the rDNA ITS region of the Jeonbuk isolate and other Didymella bryoniae from BLAST searches of GenBank was 100% and was 95.0% within the group. Nucleotide sequences of the rDNA ITS region from pure culture ranged from 98.2% to 99.8%. Phylogenetic analysis with related species of D. bryoniae revealed that D. bryoniae is a monophyletic group distinguishable from other Didymella spp., including Ascochyta pinodes, Mycosphaerella pinodes, M. zeae-maydis, D. pinodes, D. applanata, D. exigua, D. rabiei, D. lentis, D. fabae, and D. vitalbina. Phylogenetic analysis, based on rDNA ITS sequence, clearly distinguished D. bryoniae and Didymella spp. from the 10 other species studied. This study identified the Jeonbuk isolate to be D. bryoniae.

Isolation and Identification of Mushroom Pathogens from Agrocybe aegerita.

Agrocybe aegerita is an important mushroom cultivated in Korea, with good feel and a peculiar fragrance. A. aegerita can be cultivated throughout the year using culture bottles but is more susceptible to contamination than other mushrooms. Twenty-two pathogens were isolated from the fruiting bodies and compost of A. aegerita, and seven isolates were isolated from Pleurotus ostreatus to compare with the A. aegerita isolates, collected from Gimje, Iksan, Gunsan of Chonbuk, and Chilgok of Gyeongbuk Province in 2009. These isolates were identified based on morphological and molecular characteristics. Of the 29 isolates, 26 were identified as Trichoderma spp. and the remaining three were Aspergillus spp., Mucor spp., and Penicillium spp. A phylogenetic analysis revealed that the 26 isolates of Trichoderma were divided into four taxa, namely T. harzianum, T. pleuroticola, T. longibrachiatum, and T. atroviride. Among the Trichoderma spp., 16 isolates (55.2%) were identified as T. harzianum, six as T. pleuroticola (20.7%), two as T. longibrachiatum, and the remaining two were T. atroviride.

Molecular analysis of Korean isolate of barley yellow mosaic virus.

The complete sequences of both RNAs of an isolate of barley yellow mosaic virus (BaYMV) from Haenam, Korea, were determined. RNA1 is 7639 nucleotides long [excluding the 3'-poly(A)], and codes for a 270 kDa polyprotein of 2411 amino acids which contains the capsid protein (CP) at the C terminus and seven putative non-structural proteins. RNA2 is 3582 nucleotides long and codes for a polyprotein of 890 amino acids, which contains a 28 kDa putative proteinase (P1) and a 73 kDa polypeptide (P2). The whole sequences of Korean isolate (BaYMV-K) closely resembled those of an isolate from Japan (BaYMV-J) (99.6 identical nucleotides for RNA1; 99.4 for RNA2) and china (BaYMV-C) (96.7 and 96.2%, respectively) than from Germany (BaYMV-G) (93.6 and 90.4%, respectively). The greatest differences between the BaYMV-K and BaYMV-J isolates were in the 3'-NCRs of RNA1 and 5' NCRs of RNA2 and there were also some other regions of difference in Nib Pro (RNA1) and P1 (RNA2). Further, the phylogenetic analysis of CP region showed that Asian and European isolates formed distinct clusters. However, molecular variations between isolates could not be linked to earlier results showing differences in cultivar response.

Influence of plant growth promoting bacteria and Cr6+ on the growth of Indian mustard.

The Cr(6+) resistant plant growth promoting bacteria (PGPB), Pseudomonas sp. PsA4 and Bacillus sp. Ba32 were isolated from heavy metal contaminated soils and their plant growth promoting activity on the Indian mustard (Brassica juncea) were assessed with different concentrations of Cr(6+) in soil. Production of siderophores and the solubilization of phosphate were observed in both strains, PsA4 and Ba32. Production of IAA was only observed in strain PsA4. Inoculation of PsA4 or Ba32 promoted the growth of plants at 95.3 and 198.3 microg of Cr(6+)g(-1) soil. The maximum growth was observed in the plants inoculated with strain PsA4. Both strains, PsA4 and Ba32 did not influence the quantity of accumulation of chromium in root and shoot system. The present observations showed that the strains PsA4 and Ba32 protect the plants against the inhibitory effects of chromium, probably due to the production of IAA, siderophores and solubilization of phosphate.

Characterization of a novel Cr6+ reducing Pseudomonas sp. with plant growth-promoting potential.

The isolate RNP4 obtained from a long-term tannery waste contaminated soil was characterized and presumptively identified as Pseudomonas sp. The strain RNP4 tolerated concentrations up to 450 mg Cr(6+)/L on a Luria-Bartani (LB) agar medium and reduced a substantial amount of Cr(6+) to Cr(3+) in the LB liquid medium. The ability of performing multifarious activities in tandem suggested the uniqueness of isolate RNP4. The strain produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. The strain also exhibited the production of siderophore and solubilization of phosphorus in mineral salt medium and SRS1 medium, respectively. Concurrent production of IAA and siderophore and the solubilization of phosphorus revealed its plant growth promotion potential. Furthermore, the strain was able to promote the growth of black gram, Indian mustard, and pearl millet in the presence of Cr(6+). Thus, the innate capability of this novel isolate for parallel bioremediation and plant growth promotion has significance in the management of environmental and agricultural problems.

Screening of bacterial antagonists for biological control of Phytophthora blight of pepper.

The aim of this study was to assess the potential of bacterial antagonists to control Phytophthora blight of pepper caused by P. capsici using different screening methods. Among a collection of fluorescent pseudomonas isolated from the rhizosphere of pepper, twelve isolates were initially selected based on dual culture assay on potato dextrose agar and corn meal agar. Further, these twelve isolates were screened for the reduction of disease severity caused by P. capsici using detached leaves and seedling assay. Most of the antagonists showed varying levels of antagonism against P. capsici in both detached leaves and seedlings assay. In addition, few isolates increased shoot and root length of pepper in seedling assays. Among them, isolate PS119 showing highest ability to reduce the disease severity in the in vitro seedling assay was found to be the most efficient antagonists against P. capsici in the in vivo biological control tests. These results indicate that the in vitro seedling assay can be used as a rapid and more accurate technique for the selection of promising biocontrol agents against P. capsici.

Growth of Brassica juncea under chromium stress: influence of siderophores and indole 3 acetic acid producing rhizosphere bacteria.

Plant growth promoting rhizobacterial (PGPR) strains A3 and S32 have been shown to promote the growth of Brassica juncea under chromium stress which has been related to the microbial production of siderophores and indole 3 acetic acid (IAA). The aim of the present study is to evaluate the importance of siderophores and IAA producing PGPR on the growth of Brassica juncea under chromium stress. The production of IAA and siderophores were observed in the strains A3 and S32, respectively. Both PGPR strains promote the growth of Brassica juncea under chromium stress. The maximum growth was observed in plants inoculated with siderophores producing strain 32. Both the bacterial inoculum did not influence the uptake of chromium by plants. The present observation showed that PGPR isolates A3 and S32 are capable of protecting the plants against the inhibitory effects of chromium by producing the siderophores and IAA.

Growth responses of Cassia obtusifolia toward human intestinal bacteria.

1,2-Dihydroxyanthraquinone (1) was isolated from the seed of Cassia obtusifolia through bioassay-guided fractionation. 1,2-Dihydroxyanthraquinone strongly inhibited the growth of Clostridium perfringens and Escherichia coli. Structure-activity relationship revealed that 1,4-dihydroxyanthraquinone (2) and 1,8-dihydroxyanthraquinone (3) had strong growth-inhibition against C. perfringens. In growth-promoting activity, 1,2-, 1,4-, and 1,8-dihydroxyanthraquinones exhibited strong growth-promoting activity to Bifidobacterium bifidum.