Sang Gyu Kim

Silicon-Induced Cell Wall Fortification of Rice Leaves: A Possible Cellular Mechanism of Enhanced Host Resistance to Blast

ABSTRACT Locations of silicon accumulation in rice leaves and its possible association with resistance to rice blast were investigated by electron microscopy and X-ray microanalysis. A blast-susceptible cultivar, Jinmi, and a partially resistant cultivar, Hwaseong, were grown under a hydroponic culture system with modified Yoshidas nutrient solution containing 0, 50, 100, and 200 ppm of silicon. Electron-dense silicon layers were frequently found beneath the cuticle in epidermal cell walls of silicon-treated plants. Increasing levels of silicon were detected in the outer regions of epidermal cell walls. Silicon was present mainly in epidermal cell walls, middle lamellae, and intercellular spaces within subepidermal tissues. Furthermore, silicon was prevalent throughout the leaf surface, with relatively small deposition on stomatal guard cells in silicon-treated plants. Silicon accumulation and epidermal cell wall thickness in leaves were greater in cv. Jinmi than in cv. Hwaseong. However, the thickness ratios of the silicon layers to epidermal cell walls were greater in cv. Hwaseong (53.25 to 93.28%) than in cv. Jinmi (36.58 to 66.54%). Leaf blast severity was lower in cv. Hwaseong than in cv. Jinmi and was significantly reduced in silicon-treated plants of both cultivars. These results suggest that silicon-induced cell wall fortification of rice leaves may be closely associated with enhanced host resistance to blast.

A novel gene, ROA, is required for normal morphogenesis and discharge of ascospores in Gibberella zeae.

ABSTRACT Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.

Biocontrol Efficacies of Bacillus Species Against Cylindrocarpon destructans Causing Ginseng Root Rot

Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921,Korea(Received on July 19, 2011; Revised on October 4, 2011; Accepted on October 4, 2011)Two antifungal bacteria were selected from forest soilsduring the screening of microorganisms antagonistic toCylindrocarpon destructans, a cause of ginseng root rot.The antifungal bacteria were identified as Bacillussubtilis (I4) and B. amyloliquefaciens (yD16) based onphysiological and cultural characteristics, the Biologprogram, and 16S rRNA gene sequencing analyses.Antagonistic activity of both bacterial isolates to C.destructans increased with increasing temperature.More rapid starch hydrolytic activity of the bacteriawas seen on starch agar at higher temperatures than atlower temperatures, and in the higher density inoculumtreatment than in the lower density inoculum treatment.The bacterial isolates failed to colonize ginseng root theroot tissues inoculated with the bacteria alone at aninoculum density of 1 × 10

Comparison of Microbial Fungicides in Antagonistic Activities Related to the Biological Control of Phytophthora Blight in Chili Pepper Caused by Phytophthora capsici

Department of Plant Medicine, Sunchon National University, Suncheon 540-742, Korea (Received on August 3, 2010; Accepted on October 10, 2010)Two similar microbial fungicides (termed as MA andMB) developed in a Korean biopesticide company wereanalyzed and compared each other in their biocontrolactivities against the phytophthora blight of chili peppercaused by Phytophthora capsici. MA and MB containedthe microbe Paenibacillus polymyxa and Bacillus sub-tilis, respectively, with concentrations over those postedon the microbial products. In comparison of the isolatedmicrobes (termed as MAP from MA and MBB fromMB) in the antagonistic activities against P. capsici waseffective, prominently against zoospore germination,while MBB only significantly inhibited the myceliagrowth of the pathogen. Some effectiveness of MAP andMBB was noted in the inhibition of zoosporangiumformation and zoospore release from zoosporangia;however, no such large difference between MAP andMBB was noted. In a pot experiment, MA reduced theseverity of the phytophthora blight more than MB,suggesting that the disease control efficacy would bemore attributable to the inhibition of zoospore germi-nation than mycelia growth of P. capsici. These resultsalso suggest that the similar microbes MA and MBtargeting different points in the life cycle of the patho-gen differ in the disease control efficacies. Therefore,to develop microbial fungicides it is required to ex-amine the targeting points in the pathogen’s life cycleas well as the action mode of antagonistic micro-organisms.Keywords : biocontrol, chili pepper, microbial fungicides,Phytophthora capsiciPhytophthora blight of chili pepper caused by Phytophthoracapsici is a serious disease in the crop cultivation, of whichthe main control measures include fungicide spray andcultural practices such as crop rotation (Hwang, 2002;Hwang and Kim, 1995; Ristano and Johnston, 1999). Severalfungicides including metalaxyl, oxadixyl, propamocarb,copper oxychloride, chlorothalonil, dithianone, etc. areavailable, but it is very difficult for them to exert theirefficacy in controlling the disease because of the soilbornenature and exceedingly rapid spread of the disease (Hwang,2002; Kim, 2004). Crop rotation also is not capable ofexerting sufficiently effective controlling of the diseasebecause the pathogen survives as oospores (primary inoculum)in soil without host plants for several years (Hwang andKim, 1995). It is necessary to develop alternative strategiesfor the control of the phytophthora blight of chili pepper.Use of antagonistic microbes as biological control agents(BCAs) is suggested as a good way to cope with this kindof disease problem (Hwang, 2002). Over a hundred microbial products are currently register-ed or marketed as BCAs worldwide (Whipps and McQuiken,2009), and presently a total of 14 microbial fungicides arecommercially registered in Korea. However, inconsistencyin the efficacy of BCAs in large-scale glasshouse or fieldconditions is one of major constraints in biological diseasecontrol (Whipps and McQuiken, 2009). This kind of pro-blem may also hold true to the above microbial fungicidesmarketed in Korea, especially those for targeting soil-bornepathogens, for which crop growers are not convinced oftheir practical usage in large-scale crop plantations. There-fore, every possible way should be sought to increase effi-cacy and consistency of BCAs even after their commerciali-zation in the market. Selection of good antagonists and their characterizationsuch as mode of actions are important processes and criteriafor the successful development of BCAs (Whipps andDavies, 2000). Especially the understanding on the actionmode (mechanism) of BCAs in the biological control mayprovide crucial information that can be used for practicalapplications of BCAs, promoting their consistent controlefficacy. In this study, two similar microbial fungicides re-gistered and marketed in Korea (both targeting Phytophthoraspp.) were compared in antagonistic mechanisms and bio-logical control efficacies against the phytophthora blight ofchili pepper caused by P. capsici to examine their relationsin the biological control.

First Report on Bacterial Soft Rot of Graft-cactus Chamaecereus silvestrii Caused by Pectobacterium carotovorum subsp. carotovorum in Korea

A soft stem rot disease was observed on Chamaecereus silvestrii (Korean name: Sanchui), a scion of graft-cactus, in major growing areas of Suwon (National Horticulture Research Institute), Anseong, Eumseong, Cheonan, Daegu, and Goyang, Korea during 2000 and 2001. Typical symptoms were soft rots characterized by moist and watery decay of the whole cactus stem, which initiated as small water-soaked lesions and enlarged rapidly to the entire stem. The causal organism isolated from the infected stems was identified as Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora subsp. carotovora) based on its physiological and biochemical characteristics and confirmed by the cellular fatty acid composition and Biolog analyses. Artificial inoculation of the bacterium produced the same soft rot symptoms on the cactus stems, from which the same bacterium was isolated and identified. This is the first report of the P. carotovorum subsp. carotovorum in the graft-cactus C. silvestrii in Korea.

First Description of Crown Gall Disease on Ginseng

In March of 2003, tumors (galls) were observed on ginseng seedling roots in ginseng seedbeds at Yeoju, Gyeonggi province, Korea. Symptoms were spherical or galls with about 0.5-1.0 cm in diameter formed on the upper through middle parts of the primary roots. Bacterial isolates obtained from the root galls were Gram-negative, rod-shaped with peritrichous flagella, aerobic, not forming yellow or orange colonies on nutrient glucose agar, yeast extract-dextrose CaCO3 agar and nutrient-broth yeast extract agar, nonfluorescent on King`s B agar, and non-spore forming, which were identical to characteristics of the genus Agrobacterium. They were identified as Agrobacterium tumefaciens with 0.732-0.993 similarities in 100% probability by the Biolog analyses. The 16S rRNA gene partial sequences of the six isolates tested (Genbank Accession EF486308~EF486313) were 100% homologous to those of other A. tumefaciens strains (GenBank accession AF501343, AY701900, AY701898, AY701899). The above results confirmed that this bacterium is A. tumefaciens. Pathogenicity of the bacteria was proved by the inoculation test on carrot root discs and tomato seedlings. This is the first description of A. tumefaciens causing root gall in ginseng seedling. The disease occurred locally and sparsely, but considering its appearances in seedbeds suggests that the ginseng root gall may become a threat to ginseng in Korea.

Effect of Delayed Inoculation After Wounding on the Development of Anthracnose Disease Caused by Colletotrichum acutatum on Chili Pepper Fruit

Detached chili pepper fruits were inoculated with the conidial suspension of Colletotrichum acutatum JC-24 simultaneously (simultaneous inoculation, SI) and at delayed time (delayed inoculation, DI) after wounding with (delayed wound inoculation, DWI) or without additional wounding (delayed non-wound inoculation, DNI) at the inoculation time. Disease severity was significantly lowered by DNI, compared to SI. By DNI, the disease reduction rates were proportional with the length of delayed time, and greater at the high temperature range (18, 23 and ) than at the low temperature () tested. DWI was also effective in reducing the disease severity especially at 18oC; however, its effectiveness was lower than for DNI. In light microscopy, parenchyma cells at the wounding sites were modified structurally, initially forming new cell walls crossing cytoplasm, enlarged with multiple periclinal cell divisions, and finally layered like wound periderms. In DWI, the above structural modifications occurred, showing the restriction of the fungal invasion by the cell walls in enlarged modified cells, while no definite cellular modifications were found with proliferation of fungal hyphae in SI. Sclerenchyma-like cells with thickened cell walls were proliferated around the wounding sites, which were partially dissolved by DWI, probably leading to some disease development. All of these results suggest that the decline of the anthracnose disease in pepper fruit by the delayed inoculations may be derived from the structural modifications related to the healing processes of the previous wound inflicted on the tissues.

Biological and Structural Mechanisms of Disease Development and Resistance in Chili Pepper Infected with the Root-knot Nematode

Biological and structural mechanisms of the nematode disease development in chili pepper caused by the rootknot nematode, Meloidogyne incognita, were investigated. Out of 39 chili pepper cultivars/lines tested, six were found resistant, while 33 were susceptible to M. incognita, of which a susceptible cultivar Chilseongcho and three resistant cultivar/lines CM334, 02G132 and 03G53 with different resistance degrees were selected for microscopic studies on the disease development. Gall formation was greatly reduced in the resistant cultivars/ lines. Nematode penetration occurred both in the susceptible and resistant chili pepper roots; however, the penetration rates were significantly lowered in the three resistant peppers compared to the susceptible pepper cv. Chilseongcho. In the susceptible pepper, giant cells were extensively formed with no discernible necrosis around the nematode feeding sites. In the highly resistant pepper cultivar CM334, no giant cell was formed, but extensive necrosis formation was observed around the penetrating nematodes. In the other two resistant pepper lines (02G132 and 03G53), both giant cells and prominent necroses were formed, and the necrotic responses appeared to inhibit the further development of giant cells or accelerate their early degeneration. Although the nematode penetration was retarded significantly in the resistant cultivar/lines, all of the above results suggest that the disease resistance of pepper may be related to post-infectional defense mechanisms (nematode growth and development) more than pre-infectional ones (penetration and establishment). Variations in structural modifications in the resistant cultivar/lines may reflect their genetic differences related to the nematode resistance.

Different Structural Modifications Associated with Development of Ginseng Root Rot Caused by Cylindrocarpon destructans

Root rot caused by Cylindrocarpon destructans is one of the most important diseases of ginseng (Panax ginseng C. A. Meyer). Two types of symptoms found in ginseng root rot are black root rot and rusty root (rusty spots), in which disease severities are high and low, respectively. Symptom development and related histopathological changes were examined in an inoculation test on 2-year-old ginseng roots using virulent (Cy9801) and avirulent (Cy0001) isolates of C. destructans under different temperature conditions (13, 18, 23, and 28oC). Black root rot was only induced by Cy9801 in the lower temperature range (13, 18, and 23oC) and not at the higher temperature (28oC). No black root rot, but only rusty spot symptoms, were induced by Cy0001 at all temperatures tested except 13oC, at which no symptoms occurred on over half of inoculation sites, suggesting disease development was influenced by pathogen virulence and temperature. Wound periderms were formed in all root tissues with rust spot symptoms at 28oC caused by Cy9801 and at 18, 23, and 28oC temperatures caused by Cy0001. No wound periderm was formed at 13oC by either Cy9801 or Cy0001. Light microscopy revealed that the wound periderm was formed by initial cell divisions in cell wall formation and/or additional cell wall layering in parenchyma cells without obvious nuclear division, followed by layering of the divided cells adjacent to the inoculation sites, blocking the spread of the rot. These results suggest that disease development declined at lower temperatures and by the formation of a wound periderm at higher temperatures, and that ginseng rusty root may develop under conditions unfavorable for further disease development of C. destructans.

First Report of Black Spot Caused by Alternaria alternata on Grafted Cactus

Chemical Biotechnology Research Center, KRICT, Daejeon 305-605, Korea(Received on June 25, 2009; Accepted on November 25, 2009)A stem spot disease was observed on Gymnocalyciummihanovichii (Korean name: Bimoran), a scion of graft-cactus, in major growing areas of Goyang and Eum-seong, Korea during 2008 and 2009. Typical symptomswere initial blackish brown lesions produced mainly onareoles and scars of injured stem tissues, graduallybecoming large black spots. A causal organism isolateCD2-7A isolated from the infected stems was identifiedas Alternaria alternata based on its morphologicalcharacteristics and confirmed by the DNA sequencinganalysis of ITS, glyceraldehydes-3-phosphate dehydro-genase (gpd), and allergen Alt a1 (Alt a 1) genes.Artificial inoculation of the fungal isolate CD2-7Aproduced the same rot symptoms on the cactus stems,from which the same fungus was isolated and identified.This is the first report of the black spot caused by A.alternata in the grafted cactus.Keywords : Alternaria alternata, graft-cactus, Gymnocaly-cium mihanovichii, pathogenicityMost cactuses are stem succulents, having fleshy stems. Agrafted cactus, which is composed of stock and scioncactuses, is a major commercial product in Korea and oneof the most important exporting ornamental plants due to itshigh quality in the world market. The most widely culti-vated stock cactus is a three-angled cactus (Hylocereustrigonus), while two scion cactuses commonly used inKorea are Gymnocalycium mihanovichii and Chamaecereussilvestrii, of which the Korean common names are Bimoranand Sanchui, respectively. G. mihanovichii is more popular-ly cultivated in Korea than C. silvestrii.One of the limiting factors for cultivating grafted cactusesin greenhouses is due to stem rot diseases especially causedby fungi. They include stem rots caused by Fusariumoxysporum (Chang et al., 1998), Bipolaris cactivora (Hyunet al., 1998; Kim et al., 2004), and Glomerella cingulata(Kim et al., 2000). In our surveys of cactus diseases during2008 and 2009, however, we often observed some stemdiseases of G. mihanovichii with no fungi belonging to theabove reported genera. Therefore, we report the occurrence of a fungal stemdisease of G. mihanovichii and the identification andcharacterization of the fungal pathogen.Disease occurrence and symptoms. Occurrence of a stemdisease was observed frequently on G. Mihanovichii inmajor growing areas of Goyang and Eumseong, Koreaduring 2008 and 2009. Symptoms were characterized byblackish brown lesions produced mainly on areoles andscars of injured stem tissues (Fig. 1A, B). Sometimes largeportions of the cactus stems were diseased with large blackspots at advanced infection stages. Fungal isolation and pathogenicity. Pathogen isolationwas conducted using infected G. mihanovichii stems. Smallstem tissues were cut from the edge of the diseased areawith flame-sterilized razor blade and surface-sterilized with75% ethanol for 30 sec and 1% sodium hypochlorite for 60sec, and rinsed with sterile water. The stem tissues wereplaced on potato-dextrose agar (PDA) supplemented with0.1% lactic acid, incubating at 25