Mycelial Colonies Research Articles

Expression of a synthetic antimicrobial peptide, D4E1, in Gladiolus plants for resistance to Fusarium oxysporum f. sp. gladioli

The main pathogen of Gladiolus plants is Fusarium oxysporum, a soilborne fungus that infects roots and corms resulting in death of the plant. Purified D4E1, a synthetic antimicrobial peptide, was previously reported (De Lucca et al. 1998) to inhibit F. oxysporum spores from forming mycelial colonies in vitro at a concentration of 3 μM making it a candidate gene for genetic engineering of Gladiolus for resistance to F. oxysporum. Gladiolus cv. Peter Pears plants were transformed by particle bombardment with plasmid DNA containing a 90 bp D4E1 gene that was under the control of the duplicated CaMV 35S promoter. Five of the 14 independently transformed plant lines were evaluated for resistance to F. oxysporum. Transgenic plants were tested in vitro for resistance to F. oxysporum, and several lines appeared to be more resistant than the control plants that lacked D4E1. Cell extracts of transgenic Gladiolus lines 6(1) and 7(1) inhibited germinated spores of F. oxysporum f. sp. gladioli from forming mycelial colonies by 34 and 38 %, respectively, in vitro. F. oxysporum f. sp. gladioli was transformed with the ECFP (cyan) gene allowing us to follow the growth of F. oxysporum during infection of D4E1-transformed and non-transformed roots. Fluorescence observations using confocal laser scanning microscopy showed that 3–10 days after infection, F. oxysporum covered the surface of the root and formed pseudo-appressoria, but hyphae were never observed to penetrate cells of the root. Ten days after infection with F. oxysporum, non-transformed roots had completely disintegrated whereas transgenic roots of line 7(1) were just beginning to lose their cellular integrity. Cell extracts from the five transgenic lines showed either an inhibition of F. oxysporum mycelial colony formation or less fungal hyphae were observed to infect their roots as compared to non-transformed Gladiolus plants.

Caracterização cultural e patogenicidade de isolados de Lasiodiplodia theobromae em plantas de cajaraneira

In recent years, Lasiodiplodia theobromae, has become an important pathogenic agent to many crops. It has been widely spread in all tropical and subtropical regions of the world. The increasing extension of diseases caused by L. theobromae in tropical fruits has caused enormous losses. Therefore, the need for basic knowledge about this pathogen is a must. This study aimed to characterize isolates of L. theobromae, evaluating the cultural aspects and pathogenicity in seedlings of Spondias cytherea in Northeast Brazil. The mycelial growth, colony morphology and pathogenicity of isolates on S. cytherea were evaluated. The results showed variation in mycelial growth and pathogenicity of L. theobromae in S. cytherea. It was also observed that there is a positive interaction between the mycelial growth in culture media and the aggressiveness of L. theobromae isolates while infecting the seedlings.

Characterization of the Maize Stalk Rot Pathogens Fusarium subglutinans and F. temperatum and the Effect of Fungicides on Their Mycelial Growth and Colony Formation.

Maize is a socioeconomically important crop in many countries. Recently, a high incidence of stalk rot disease has been reported in several maize fields in Gangwon province. In this report, we show that maize stalk rot is associated with the fungal pathogens Fusarium subglutinans and F. temperatum. Since no fungicides are available to control these pathogens on maize plants, we selected six fungicides (tebuconazole, difenoconazole, fluquinconazole, azoxystrobin, prochloraz and kresoxim-methyl) and examined their effectiveness against the two pathogens. The in vitro antifungal effects of the six fungicides on mycelial growth and colony formation were investigated. Based on the inhibition of mycelial growth, the most toxic fungicide was tebuconazole with 50% effective concentrations (EC50) of <0.1 μg/ml and EC90 values of 0.9 μg/ml for both pathogens, while the least toxic fungicide was azoxystrobin with EC50 values of 0.7 and 0.5 μg/ml for F. subglutinans and F. temperatum, respectively, and EC90 values of >3,000 μg/ml for both pathogens. Based on the inhibition of colony formation by the two pathogens, kresoxim-methyl was the most toxic fungicide with complete inhibition of colony formation at concentrations of 0.1 and 0.01 μg/ml for F. subglutinans and F. temperatum, respectively, whereas azoxystrobin was the least toxic fungicide with complete inhibition of colony formation at concentrations >3,000 μg/ml for both pathogens.

First Report of Powdery Mildew Caused by Blumeria graminis on Festuca arundinacea in China.

Tall fescue (Festuca arundinacea Schreb), a predominant cool-season perennial grass, is widely used as forage and turf grasses in China. In July 2013, powdery mildew was observed on 10 F. arundinacea lawns (about 0.5 ha in total) in Urumchi, Xinjiang Province, China, with 20 to 30% of the area being infected. Signs of the disease initially appeared as irregular white mycelial colonies on the adaxial surface of infected leaves. As the disease progressed, the colonies covered the whole adaxial surface and white patches appeared on the abaxial surface of infected leaves. Conidiophores were unbranched and cylindrical with swollen bases, measuring 13.3 to 15 × 16.7 to 20 μm, and borne vertically on hyphae. Each conidiophore produced 10 to 18 conidia in a chain. The conidia were oval, one-celled, and colorless, measuring 8.1 to 9.8 × 26 to 29.7 μm. Cleistothecia were black, spherical, and 164.3 to 207.3 μm in diameter, each of which contained 9 to 26 asci. Asci were oblong or ovate, measuring 32.1 to 40 × 85.7 to 96.4 μm. Asci were petiolate, containing eight ascospores. Ascospores were round to oval, colorless, one-celled, measuring 19.1 to 22.5 × 11.7 to 13.6 μm. Based on morphological characteristics of the anamorph and the teleomorph, the fungus was identified as Blumeria graminis (DC.) Speer. Additionally, the internal transcribed spacer (ITS) of 563 bp was amplified from DNA of conidia using ITS1 and ITS4 primers (4). The ITS sequence was deposited in GenBank (Accession No. KF545644). The ITS sequence showed 100% homogeneity with those of B. graminis on Poa pratensis in Swizerland (AB273540) and on P. bulbosa in Iran (AB273551) (1), which further confirmed the identification. Ten 3-week-old healthy plants were inoculated by spraying a spore suspension (1 × 105 conidia ml-1) made from conidia brushed from infected plants, and 10 plants sprayed with sterile distilled water were served as controls. All the plants were placed in the same growth chamber at 20°C, 80% humidity, and 16-h photoperiod. Twenty days after inoculation, typical signs and symptoms of powdery mildew were observed on all the inoculated plants, whereas no symptoms were observed on the controls. Microscopic and ITS analysis showed that the fungus on the inoculated plants is identical to that on diseased field plants. B. graminis on F. arundinacea has been observed in a few European countries (1), Israel (3), and the United States (2). To our knowledge, this is the first report of powdery mildew caused by B. graminis on F. arundinacea in China, which will increase the difficulty to prevent powdery mildew on grasses including cereals.

First Report of Leaf and Sheath Spot Caused by Waitea circinata var. zeae on Paspalum vaginatum and Zoysia tenuifolia in China.

Waitea circinata var. zeae was previously reported as the causal agent of leaf and sheath spot on Festuca arundinacea (1) and Panicum tennesseense (2) in the United States. In late May to mid-September 2013, a disease resembling leaf and sheath spot was observed on Paspalum vaginatum in fairways from several golf courses in Hainan Province, China. Affected plants initially had large yellow and brown spots on leaves and sheathes, then the whole plant turned yellowish-brown and eventually died. The same symptoms were also observed on a lawn established with Zoysia tenuifolia in Hainan University. Symptomatic leaves were surface sterilized in 1% hypochlorite for 1 min, rinsed with sterile water three times, and plated onto potato dextrose agar (PDA) amended with 0.01% gentamicin sulfate. Two Rhizoctonia-like fungal isolates were obtained from the diseased P. vaginatum (Isolate no. ML-WC1) and Z. tenuifolia (Isolate no. HNU-1) samples. After incubation on PDA for 1 week at 25°C, white mycelial colonies developed and eventually turned a salmon color with age. Small, white, spherical sclerotia (0.5 to 1 mm in diameter) were observed submerged throughout the agar media after incubation for 1 week and turned a reddish-brown color within 4 weeks. The two isolates were tentatively identified as W. circinata var. zeae based on these characteristics. The internal transcribed spacer (ITS) region of the ribosomal DNA gene was amplified and sequenced using primer pair ITS1/ITS4. The sequences of the two isolates (GenBank Accession Nos. KJ717943 and KJ717944) were more than 99% similar to W. circinata var. zeae (JQ688056 and JQ688059) sequences deposited in GenBank. To confirm pathogenicity, inocula were prepared by incubating autoclaved rye grains with strains ML-WC1 and HNU-1 for 2 weeks at 25°C. Ten colonized rye grains were uniformly spread around the crowns of 6-week-old P. vaginatum and Z. tenuifolia seedlings grown in 10-cm-diameter pots. Each isolate was placed in four separate pots and four control plants were treated with non-inoculated grain. All pots were covered with translucent plastic bags and placed in a greenhouse at 24 ± 2°C with a 12-h light/dark cycle. By 1 week post-inoculation, significant blighting of leaves and sheaths was observed, while non-inoculated plants showed no symptoms. W. circinata var. zeae was successfully re-isolated from symptomatic plants and confirmed by its Rhizoctonia-like mycelium and small, reddish-brown, spherical sclerotia on the PDA. Recently a related species, W. circinata var. circinata, causing brown ring patch on two cool-season grasses was reported in China (3). However, the isolates of W. circinata var. zeae were distinguished from W. circinata var. circinata base on ITS sequence data and morphological characters. To our knowledge, this is the first report of W. circinata var. zeae infecting P. vaginatum and Z. tenuifolia in China.

Alternaria brassicicola Causes a Leaf Spot on Isatis indigotica in China.

Chinese woad (Isatis indigotica) is a biennial herb in the Brassicaceae that is widely cultivated in China. Extracts from the roots and leaves have potential pharmaceutical use for treatment of flu, encephalitis, measles, hepatitis, and mumps (2). In June 2012, a leaf spot was observed on 1-year-old plants of I. indigotica in the medicinal garden of Jilin Agricultural University, Changchun, Jilin Province, China. More than 50% of the leaves and 100% of the plants in the garden were symptomatic. In the initial stage of infection, irregular to circular, dark gray spots, each surrounded by a chlorotic halo, appeared on leaves. The spots ranged from pinpoint to 5 mm in diameter. Some spots enlarged and coalesced, forming concentric rings. Black, sunken, fusiform lesions were observed on the petioles. Lesions gradually dried and exhibited a shot-hole appearance, and entire infected leaves desiccated. Small pieces of infected leaves and petioles were surface-disinfested in 75% ethanol for 60 s, rinsed thrice in sterilized distilled water, dried, and plated on potato dextrose agar. Olive-green mycelium developed after 2 days of incubation at 25°C, turned dark green, and covered the petri dish 10 days later. The periphery of each colony was gray and velvety. On potato carrot agar medium, conidia formed on branched chains. Conidiophores arose singly or in clusters, were straight or flexuous, separated, and measured 6.8 to 26.7 × 3.1 to 11.9 μm Conidia on host plant tissues were olivaceous, cylindrical or inverted clavate, and 25.8 to 65.2 × 10.9 to 18.3 μm Larger conidia were cylindrical or obclavate, and smaller conidia were oval. Transverse and longitudinal septa of conidia ranged from 3 to 10 and from 0 to 7 μm, respectively. A very small conidial beak or no beak was observed on each conidium. On the basis of these morphological characteristics,the fungus was identified as Alternaria brassicicola (3). A PCR assay with the ITS4 and ITS5 primers was used to amplify DNA extracted from each of four isolates (1). The sequence (567 bp) of isolate Sl-8 was submitted to GenBank (Accession No. KF531832), and showed 100% similarity to that of an A. brassicicola isolate (AF392985.1), confirming the species identification. Pathogenicity assays with 10 single-conidium isolates were done by spraying a conidial suspension (1 × 106 conidia/ml) of each isolate, or sterilized water for the control treatment, onto healthy leaves and petioles of five 3-month-old plants of I. indigotica. Inoculated and control plants were enclosed in plastic bags for 48 h. After 7 days, symptoms on inoculated plants were similar to those on the original diseased plants, while control plants remained symptomless. Re-isolation from inoculated plants produced mycelial colonies with morphological characteristics of A. brassicicola, fulfilling Koch's postulates. No fungus was isolated from control plants. A. napiformis and A. brassicae have been reported as causal agents of Alternaria leaf spot on I. indigotica in China (3). To our knowledge, however, this is the first report of A. brassicicola as a pathogen on I. indigotica in China.

Identification and characterization of pathogenic Pestalotiopsis species to pecan tree in Brazil

The objective of this work was to characterize and cluster isolates of Pestalotiopsis species and to identify those that are pathogenic to pecan, based on morphological and molecular characters. Pestalotiopsis spp. isolates were identified by sequencing the internal transcribed spacer (ITS) and β?tubulin regions. Identification methods were compared to indicate the key morphological characters for species characterization. Thirteen isolates were used for the pathogenicity tests. Morphological characterization was performed using the following variables: mycelial growth rate, sporulation, colony pigmentation, and conidial length and width. Ten pathogenic isolates were identified, three as -tubulin regions. Identification methods were compared to indicate the key morphological characters for species characterization. Thirteen isolates were used for the pathogenicity tests. Morphological characterization was performed using the following variables: mycelial growth rate, sporulation, colony pigmentation, and conidial length and width. Ten pathogenic isolates were identified, three as Pestalotiopsis clavispora and three as P. cocculi. The other isolates remained as an undefined species. The morphological characters were efficient for an initial separation of the isolates, which were grouped according to differences at species level, mainly colony diameter, which was identified as an important morphological describer. Beta-tubulin gene sequencing was less informative than the ITS region sequencing for species identification.

First Report of "Cylindrocarpon" pauciseptatum Associated with Black Foot Disease of Grapevine in Brazil.

Since 1999, the decline of American grapevines (Vitis labrusca L.) has been common in Rio Grande do Sul, Brazil (1). In August 2012, V. labrusca with black foot symptoms were collected in vineyards in the Serra Gaúcha Region. Symptomatic plants had low vigor, vascular lesions, delayed budding, and decline and death of vines. Symptomatic roots had necrotic lesions and reduced biomass. Fungal isolations were made from necrotic root and crown fragments (own-rooted cultivar) on potato dextrose agar (PDA) medium amended with 0.5 g L-1 streptomycin sulfate. Putative colonies of "Cylindrocarpon" pauciseptatum Schroers & Crous were obtained from single macroconidia isolations. Two isolates were used to confirm the identity of isolated colonies: Cy12UFSM and Cy13UFSM. After incubation in the dark for 10 days at 20°C, the isolated mycelial colonies, which were cottony white to felty in texture, became dark orange to brown. Both isolates produced chlamydospores in chains at 40 days. Chlamydospores of Cy12UFSM and Cy13UFSM were 9 to 12 μm and 5 to 11.5 μm in diameter. Sporodochia formation on carnation leaf agar (CLA) medium was observed after 30 days. To encourage development of conidia, the isolates were grown on spezieller nährstoffarmer agar (SNA) medium for five weeks at 20°C with addition of two pieces of 1 cm2 filter paper. Microconidia of Cy12UFSM were 4 to 8.5 × 3.5 to 5 μm and those of Cy13UFSM were 3.5 to 7.5 × 3 to 5 μm. Macroconida were predominantly 3-septate (Cy12UFSM was 36 to 45 × 7.5 to 9 μm and Cy13UFSM was 30 to 38 × 7.5 to 8 μm), but 1-, 2- septate macroconidia were observed. The sizes of the three spore types and colony morphology for our isolates were similar to those described by Schroers et al. (3) for "C." pauciseptatum. To further confirm the identity of Cy12UFSM and Cy13UFSM, multi-gene DNA sequence analysis (rDNA-ITS, β-tubulin, and histone H3) was conducted using primer pairs ITS1 and ITS4 (4), Bt2a and Bt2b, and H3-1a and H3-1b (2), which amplify the ITS1-5.8S rRNA-ITS2 genes, part of the β-tubulin gene, and the histone H3 gene, respectively. Sequences of these three regions had 99, 99, and 97% similarity with references sequences of "C." pauciseptatum (isolate Cy238; accessions ITS [JF735307]; β-tubulin [JF735435], and histone H3 [JF735582], respectively). To evaluate pathogenicity, 4-month-old rooted cuttings of V. labrusca cv. Bordô were inoculated with two isolates by immersing them in a conidial suspension (106 conidia ml-1) for 60 min. Ten single-vine replicates were used for each isolate, and 10 water-inoculated vines were included as controls. Thirty days after inoculation, vines were re-inoculated with 40 ml of a 106 conidia ml-1 suspension to ensure root infection. After 4 months, the inoculated plants had reduced root mass relative to controls (39.18% for Cy12UFSM and 18.27% for Cy13UFSM). Inoculated plants also had root and crown necrosis, vascular lesions, shoot decline, and vine mortality (60 and 80% mortality for Cy12UFSM and Cy13UFSM, respectively). All water-inoculated control plants remained symptomless. The fungi Cy12UFSM and Cy13UFSM were re-isolated from infected woody tissues, confirming Koch's postulates. To our knowledge, this is the first report of "C." pauciseptatum associated with black foot disease of grapevine in Brazil, which may potentially impact the sustainability of grapevine nurseries and vineyard productivity.

Morphological and molecular characterization of Fusarium spp pathogenic to pecan tree in Brazil.

The occurrence of Fusarium spp associated with pecan tree (Carya illinoinensis) diseases in Brazil has been observed in recent laboratory analyses in Rio Grande do Sul State. Thus, in this study, we i) obtained Fusarium isolates from plants with disease symptoms; ii) tested the pathogenicity of these Fusarium isolates to pecan; iii) characterized and grouped Fusarium isolates that were pathogenic to the pecan tree based on morphological characteristics; iv) identified Fusarium spp to the species complex level through TEF-1α sequencing; and v) compared the identification methods used in the study. Fifteen isolates collected from the inflorescences, roots, and seeds of symptomatic plants (leaf necrosis or root rot) were used for pathogenicity tests. Morphological characterization was conducted using only pathogenic isolates, for a total of 11 isolates, based on the mycelial growth rate, sporulation, colony pigmentation, and conidial length and width variables. Pathogenic isolates were grouped based on morphological characteristics, and molecular characterization was performed by sequencing TEF-1α genes. Pathogenic isolates belonging to the Fusarium chlamydosporum species complex, Fusarium graminearum species complex, Fusarium proliferatum, and Fusarium oxysporum were identified based on the TEF-1α region. Morphological characteristics were used to effectively differentiate isolates and group the isolates according to genetic similarity, particularly conidial width, which emerged as a key morphological descriptor in this study.

Utilizing Soybean Milk to Culture Soybean Pathogens

Liquid and semi-solid culture media are used to maintain and proliferate bacteria, fungi, and Oomycetes for research in microbiology and plant pathology. In this study, a comparison was made between soybean milk medium, also referred to as soymilk, and media traditionally used for culturing soybean pathogens to determine if soymilk medium was an effective medium for growth of Colletotrichum truncatum, Fusarium virguliforme, Macrophomina phaseolina, Passalora sojina, Phomopsis longicolla, Phytophthora sojae, Pythium irregulare, Rhizoctonia solani, and Sclerotinia sclerotiorum. Based on radial mycelial colony growth rates, C. sojina grew significantly (P C. truncatum and F. virguliforme grew significantly (P P S. sclerotiorum grown on SDA as compared to PDA. Soymilk used with agar or used alone as a broth may be an option for replacing more expensive processed culture media.

First Report of Vinca Blight Caused by Phytophthora nicotianae in Northwestern Mexico.

Vinca (Catharantus roseus (L.) G. Don) is a common ornamental landscape plant. From July to September 2012, blighted and wilted vinca plants were found in retail stores, commercial nurseries, and urban landscape areas of Culiacan, Sinaloa, in northwestern Mexico. In several commercial nurseries and a retail store, incidence of the unknown disease on vinca plants ranged from 20 to 50%, resulting in significant economic losses. Symptoms of the disease started with a foliar blight, and if warm and wet conditions were present, the disease progressed, causing plant wilting and death. Surface-sterilized (0.5% NaOCl 1 min) diseased plant tissue was plated on V8 agar medium, and after 72 h of incubation at 25°C, white colonies of coenocytic mycelium were developed from the plated tissues. Isolates produced cottony colonies on V8 agar medium, grew well between 7 and 30°C (optimum of 25°C), and produced spherical, intercalary, and terminal chlamydospores (17 to 30 μm) and non caducous, papillate, spherical to ovoid sporangia of 30 to 39 × 21 to 31 μm. Based on these morphological characteristics, Phytophthora isolates were identified as Phytophthora nicotianae Breda de Haan (1,3). The identity of two representative isolates OV4 and OV11 was confirmed by sequence analysis of the rDNA internal transcribed spacers (ITS; GenBank Accession Nos. KC248202 and KC248201), and of the β-tubulin (β-tub; KC248404 and KC248403) and translation elongation factor 1-α (EF1-α; KC248206 and KC248205) genes. Comparative sequence analysis against the NCBI nucleotide database showed a high degree of identity with reference sequences of P. nicotianae (ITS, 99%; β-tub, 99%; EF1-α, 100%) (2). A pathogenicity test with a representative isolate of P. nicotianae was performed on 10-week-old healthy vinca seedlings (n = 10). An aliquot of 10 ml of a zoosporic suspension (104 zoospores/ml) was sprayed onto the seedlings' leaves. An equal number of non-inoculated control seedlings were sprayed with sterile distilled water. Seedlings were maintained in a moist chamber at 25°C with 80 to 90% relative humidity and watered as needed with sterile water. Inoculated plants showed initial symptoms of foliar blight after 4 days, whereas control plants remained healthy. Ten days after inoculation, inoculated plants showed severe wilting. P. nicotianae was reisolated only from inoculated plants, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. nicotianae attacking annual vinca in northwestern Mexico.

Effect of volatiles derived from Brassica plants on the growth of Sclerotinia sclerotiorum

Secondary metabolites play an important role in plant resistance mechanisms against fungal pathogens. Glucosinolate procures of isothiocyanates exist in many species of plant families and they are specific to Brassicaceae. Release of toxic volatile compounds (isothiocyanates) by the hydrolysis of glucosinolates catalysed by myrosinase enzyme was studied on Sclerotinia sclerotiorum, the causal agent of oilseed rape stem rot. The fungal colonies were exposed to hydrolysed freeze dried powder of Brassica species including: Brassica juncea var. Cutlass, Brassica rapa varieties Parkland and Echo and Brassica napus varieties Hyola401 and RGS003. The different shoot parts of the plants, including seeds, leaves, stems, petioles and roots, as sources of glucosinolates, demonstrated significant differences in the production of toxic compounds affecting the mycelial growth of S. sclerotiorum in vitro. These inhibitions suggest that the glucosinolate–myrosinase system in Brassica species is activated and produce toxic volatiles (isothiocyanates) with different potentials. Maximum inhibition was related to B. juncea var. Cutlass. To determine the volatile compounds present in Brassica plants, the powder of their shoot parts was used for GC-MS experiments and available isothiocyanates were monitored. The results of the chromatographic studies on the seeds of B juncea var. Cutlass showed the highest peak, corresponding to the 1-propene, 3-isothiocyanates, which is Allyl ITC. The fresh part of leaves of this variety demonstrated the same peak as 1-propene, 3-ITC reflecting Allyl ITC as well. To study the effect of hydrolysis substrate variations, including oxalic acid (a pathogenicity factor of pathogen) concentrations and pH levels, the pathogen mycelial colonies were exposed to volatiles derived from seed meal of variety Cutlass. The released toxic volatile compounds inhibited significantly the radial growth of the fungus, whilst only acidic pH levels affected the system and vice versa. This shows that oxalic acid concentrations and pH levels, even at physiological rates of ambient concentration and pH, did not affect the GSL-M defence system during fungal activity in the host tissues.

Caracterização cultural, morfológica e patogênica de Lasiodiplodia theobromae associado a frutíferas tropicais

Lasiodiplodia theobromae é um fungo cosmopolita, polífago e oportunista, com reduzida especialização patogênica, capaz de infectar espécies de plantas em regiões tropicais e temperadas, causando os mais variados sintomas. Este estudo teve como objetivo caracterizar isolados de L. theobromae associados a frutíferas tropicais na região nordeste, considerando os aspectos cultural, morfológico e patogênico. Foram avaliados o crescimento micelial, coloração da colônia, dimensões dos conídios e patogenicidade dos isolados em mudas de cajazeira (Spondia mombin L.), cajueiro (Anacardium occidentale L.), gravioleira (Annona muricata L.) e umbuzeiro (Spondias tuberosa Arruda). Os dados de caracterização morfológica e cultural revelaram diversidade na população do patógeno. Alta variabilidade patogênica foi também detectada, embora não tenha sido possível observar especificidade patogênica em cajueiro. O umbuzeiro apresentou maior resistência relativa ao fungo. Os dados demonstraram também uma interação entre as características morfo-culturais e a patogenicidade dos isolados de L. theobromae.

토마토재배에 사용하는 농약과 친환경농자재가 곤충 병원성 곰팡이 Beauveria bassiana의 병원성에 미치는 영향

토마토 재배에 사용되는 농자재를 이용하여 곤충 병원성 곰팡이인 Beauveria bassiana의 병원성에 끼치는 영향을 조사하였다. Chlorothalonil 등 13종의 살균제 첨가 배지에서는 B. bassiana 균사가 전혀 생장하지 못하였고 chlorothalonil 등 12종의 살균제 첨가 배지에서 균총이 형성되지 않았다. 살충제와 친환경자재를 첨가한 모든 배지에서 균사가 생장하고 포자가 형성되었지만 대부분 배지에서 무처리와 비교했을 때 유의차를 보였다. 온실가루이에 대한 살충력은 B. bassiana 단독처리일 때보다 화학살균제(polyoxin B, mandipropamid)와 친환경농자재(sulfur) 혼합시 떨어졌으며 화학살충제(pyridaben, dinotefuran)와 식물추출물 유래 친환경농자재(pyrethrum) 혼합시에는 유의차가 없었으며 온실에서 살균제인 polyoxin B의 혼합으로 온실가루이에 대한 B. bassiana의 감염력이 떨어지는 것을 확인하였다. This study was conducted to observe the influence of chemical pesticides and environmentally friendly agricultural materials (EFAMs) used in tomato cultivation on the pathogenicity of the entomopathogenic fungus, Beauveria bassiana. B. bassiana mycelium didn't grow on PDA media containing 13 fungicides including chlorothalonil and colonies were not formed on PDA media containing 12 fungicides. B. bassiana mycelium grew and colonies were formed on all PDA media containing insecticides and EFAMs, but mycelial growth and colony formation on most PDA media were significantly inhibited compared to the control. The insecticidal activity of B. bassiana against Trialeurodes vaporariorum was decreased when fungicides (polyoxin B, mandipropamid) and EFAMs containing sulfur were added, but insecticides (pyridaben, dinotefuran) and EFAMs originated from plant extracts did not have any influence on the insecticidal activity of B. bassiana. The pathogenicity of a mixture of B. bassiana and polyoxin B against T. vaporariorum was lower than that of B. bassiana alone under greenhouse conditions.

First Report of Powdery Mildew Caused by Erysiphe sedi on Kalanchoe blossfeldiana in Korea.

Kalanchoe blossfeldiana Poelln., belonging to the Crassulaceae, is a common ornamental houseplant with many cultivars. In May 2010, powdery mildew was observed on about 50% of 3,000 potted kalanchoe 'Rose Queen' plants in plastic greenhouses located in Yongin city of central Korea. Farmers producing potted kalanchoes in Yongin region stated that powdery mildew on kalanchoes was mild without causing problems for the last several years. The disease became severe from April 2010 and caused economic losses. The economic and esthetic value was reduced by the unsightly appearance of infected plants with most being unmarketable. Damage due to powdery mildew infections on kalanchoes appeared every year. A representative specimen was deposited in the Korea University herbarium (Accession No. KUS-F24911). Mycelial colonies were white, conspicuous and epiphytic on leaves and stems. Hyphae were septate, branched, and 3 to 6 μm wide. Appressoria on the hyphae were well developed, lobed, and mostly positioned in pairs. Conidiophores were cylindrical, 70 to 145 × 7 to 11.5 μm, and composed of three to four cells. Foot-cells of conidiophores were straight, cylindrical, and 28 to 48 μm long. Conidia produced singly were variable in shape, oval to cylindrical, oval or oblong-elliptical, 30 to 55 × 14 to 24 μm, lacked distinct fibrosin bodies, and showed angular/rectangular wrinkling of outer walls. Germ tubes were produced on the perihilar position of conidia. No chasmothecia were found. The morphological characteristics were consistent with descriptions of Erysiphe sedi U. Braun (1). To confirm the identity of the causal fungus, the complete ITS region of rDNA from KUS-F24911 was amplified with primers ITS5 and P3 as described by Takamatsu et al. (4) and directly sequenced. The resulting sequence was deposited in GenBank (Accession No. JX173288). A GenBank BLAST search using the present data revealed that the ITS sequence shares 100% (552/552 bp) similarity with those of E. sedi on Sedum spp. (Accession Nos. JX173289, JX173290). Pathogenicity was confirmed through inoculation by gently pressing diseased leaves onto leaves of five healthy potted kalanchoe plants. Five non-inoculated plants served as controls. Plants were maintained in a greenhouse at 22 ± 2°C. Inoculated plants developed signs and symptoms after 7 days, whereas the control plants remained symptomless. The fungus present on the inoculated plants was morphologically identical to that originally observed on diseased plants, fulfilling Koch's postulates. E. sedi is also known to infect Kalanchoe pinnata (Lam.) Pers. (= Bryophyllum calycinum Salisb.) in Romania (1,2) and other crassulaceous plants including Sedum spectabile in North America (3). To our knowledge, this is the first report of E. sedi infections of K. blossfeldiana in Korea. This disease seems to be a serious threat to the commercial production of kalanchoe plants which are cultivated under plastic greenhouses of poor ventilation and low light levels in Korea.

Influence of Different Media Variety and Growth Regulator on Mycelial Colony Proliferation of Mushroom

An experiment was conducted at the Mushroom growth house and Tissue Culture Laboratory, Horticulture Demonstration and Training Centre (HDTC), Kewatkhali, Mymensingh during February to May, 2006 to investigate the mycelial colony proliferation of different mushroom species (Oyster (Pleurotus florida), Milky (Calocybe indica) and Button (Agaricus biporus)) in different media (viz. PDA, YPDA and MEA) and different combinations and concentrations ( 0, 1, 5, 10 and 20 ppm) of different growth regulators (IAA and NAA). As a media, the best mycelial growth of 9.34 cm was found in YPDA (Yeast Potato Dextrose Agar) at 21 DAI. As a variety, the best mycelial growth of 11.90 cm was performed by Oyster at 21 DAI. In case of combined effect of different media and variety for mycelial growth, the best growth (12.57 cm) was found in Oyster with YPDA media at 21 DAI. In case of growth regulator, the best mycelial growth of 5.50 cm was found at 21 DAI with 10 ppm IAA and 4.03 cm was found at 18 DAI with 5 ppm NAA and 5.38 cm at 21 DAI with control. In case of combined effect of IAA and NAA, the best mycelial colony proliferation (6.72 cm) was found with 10 ppm IAA + 5 ppm NAA at 21 DAI. It may be concluded that among three varieties and media, Oyster was the best variety and YPDA was the superior media for mushroom cultivation. Malt extract supplemented with 10 ppm IAA+ 5 ppm NAA as found to be the best for mycelial colony proliferation of Button mushroom DOI: http://dx.doi.org/10.3329/jesnr.v5i1.11586 J. Environ. Sci. &amp; Natural Resources, 5(1): 223 – 227, 2012

감마방사선 조사에 의한 큰느타리버섯의 돌연변이 유발

본 연구는 큰느타리버섯(Pleurotus eryngii)에 감마 방사선을 조사하여 형태적, 생리적 특성이 향상된 새로운 큰느타리버섯 품종을 개발하기 위하여 수행되었다. 큰느타리버섯에서 분리한 원형질체에 0.25-1.25 KGy의 감마 방사선을 조사하였다. 원형질체는 YPMGA 배지에서 80%의 치사율을 나타냈고 이들 중 무작위로 500개의 변이체를 선발하여 PDA 배지에 배양하였다. 그 결과, 대부분의 변이체가 대조구와 비슷한 형태와 생장률을 나타냈으며, 이 중 100개의 변이체가 대조구와 약간의 차이를 보였다. 67개 변이체에 대해 cellulase 활성과 laccase 활성을 조사하였다. 그 결과, 대체적으로 대조구와 유사한 효소 활성을 나타내었고, 5개의 변이체가 대조구에 비해 더 높은 cellulase 활성을 나타내었다. 또한 감마 방사선 조사 변이체의 유전적 변이를 조사하기 위하여 UFPF-PCR을 이용하여 다형성을 조사하였다. Gamma ray irradiation mutagenesis was employed to get variants of Pleurotus eryngii with functionally enhanced and improved characteristics. Protoplasts released from P. eryngii were treated with gamma ray radiation under 0.25-1.25 KGy. Protoplast sample that showed fatality rate of 80% at the 0.25 KGy was spreaded on YPMGA (yeast, peptone, malt-extract, glucose, agar) and 500 mycelial colonies were randomly selected from the medium. Of them, 100 mutant strains with mycelial morphology and growth rate that differ to control strain were observed on PDA. The cellulase and laccase activity of 67 gamma ray-irradiated P. eryngii isolates with morphological variation were investigated. Among these, 5 isolates were higher cellulase. In addition, the genetic variation of the mutant strains was analyzed by PCR fingerprinting.

Basidiome formation of an edible wild, putatively ectomycorrhizal fungus, Phlebopus portentosus without host plant

Phlebopus portentosus is a popular wild edible ectomycorrhizal fungus in northern Thailand. In general ectomycorrhizal fungi produce basidiomes when associated with a host plant. In this paper mycelium growth and basidiome production of P. portentosus were examined in pure culture both in vitro and in pot-culture experiments. Five mycelial strains of P. portentosus were isolated from basidiomes and used in the experiments. The mycelia grew fastest on sorghum grains supplemented with fungal-host solution. The mycelia produced sclerotia-like structures after 3 wk incubation in darkness at 30 C. All strains of P. portentosus had the ability to form primordia. The primordia were formed under lowered temperature, high humidity and a 12 h photo-period. They developed to mature basidiomes after 8–12 d in in vitro. In the pot-culture primordia were found after 28–35 d incubation in the greenhouse and mature basidiomes released basidiospores within 6–8 d. Basidiospores were germinated on fungal-host medium and formed mycelial colonies. This fungus showed an ability to produce basidiomes even 2 y after the original isolation from tissues. This research provides valuable information concerning the techniques and protocols for the large scale commercial production of P. portentosus basidiomes in the absence of a host plant.

First Report of Powdery Mildew Caused by Erysiphe betae on the Invasive Weed Dysphania ambrosioides in Korea.

Dysphania ambrosioides (L.) Mosyakin & Clemants (formerly Chenopodium ambrosioides L.), commonly known as epazote, is an herb that is native to Central America, South America, and southern Mexico. As well as in its native areas, it is used as an herb, tea, and food commodity in warm temperate to subtropical areas of Europe, the United States, and Asia. In Korea, however, this plant was accidentally introduced around the 1970s and has become widely naturalized by replacing indigenous plants and disrupting native ecosystems (3). Since 2006, powdery mildew infections of epazote have been consistently found in the southern part of Korea, including Jeju Island. Specimens (n = 8) have been deposited in the Korea University Herbarium (KUS). White mycelial and conidial growth was present mostly on leaf surfaces with sparse growth on young stems and inflorescences. Severely infected leaves were malformed. Slight purplish discoloration was present on the leaves contiguous with colony growth. Mycelial colonies were conspicuous, amphigenous, and epiphytic. Appressoria on the mycelia were lobed. Conidiophores were 110 to 200 μm long and produced conidia singly. Conidia were hyaline, oblong-elliptical, measured 30 to 48 × 13 to 18 μm, lacked fibrosin bodies, and produced germ tubes on the subterminal position. Chasmothecia were amphigenous, scattered or partly clustered, dark brown, spherical, 110 to 130 μm in diameter, and contained four to seven asci. Appendages were mycelioid, numbered 50 to 80 per chasmothecium, 0.5 to 1.5 times as long as the chasmothecial diameter, one- to three-septate, and brown at the base while becoming paler toward the tip. Asci were short stalked, 60 to 75 × 30 to 38 μm, and contained three to five spores. Ascospores were ellipsoid-ovoid with dimensions of 20 to 28 × 14 to 18 μm. On the basis of these morphological features, this fungus was identified as Erysiphe betae (Vanha) Weltzien (1). To confirm the identification, the complete internal transcribed spacer (ITS) region of rDNA from KUS-F23213 was amplified with primers ITS5 and P3 and sequenced (4). The resulting sequence of 560 bp was deposited in GenBank (Accession No. JQ041419). A GenBank BLAST search with the current data showed >99% (558 of 560 bp) similarity with the results for E. betae ex Beta vulgaris (sugar beet). Therefore, the sequence analysis verified the pathogen to be E. betae. Previous epazote infections by E. betae have been recorded in Argentina, Mexico, Romania, India, and Japan (1,2). In Taiwan, an epazote powdery mildew associated with Oidium erysiphoides f. sp. chenopodii J.M. Yen, an anamorph of E. betae, was recorded (1,2). To our knowledge, this is the first record of E. betae on epazote in Korea, and the first confirmation of epazote powdery mildew being identified as E. betae on the basis of holomorphic characteristics and ITS rDNA sequences. Our field observation suggests that the powdery mildew is acting as one of several limiting factors to suppress the expansion of this invasive weed in Korea.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Ambrosia trifida in Korea.

Ambrosia trifida L., commonly known as giant ragweed, is native to North America and was introduced to Korea in the 1970s (3). It is now widely naturalized, and since 1999, has been designated as one of 11 'harmful nonindigenous plants' by the Korean Ministry of Environment because of its adverse effects on native plants. Various strategies to eradicate this noxious weed have been tried without any success (3). In September 2009, powdery mildew infections of giant ragweed were found for the first time in Dongducheon, Korea, and specimens were isolated and deposited in the Korea University Herbarium (KUS-F24683). White mycelial and conidial growth was present mostly on adaxial leaf surfaces with sparse growth on abaxial leaf sides. Severely infected leaves were malformed. Slight purplish discoloration occurred on the leaves contiguous with colony growth. Mycelial colonies were conspicuous, amphigenous, and epiphytic with indistinct to nipple-shaped appressoria. Conidiophores were 80 to 180 μm long and produced two to five immature conidia in chains. Conidia were ellipsoid or doliiform, 28 to 38 × 16 to 24 μm, and lacked distinct fibrosin bodies. Chasmothecia were amphigenous, scattered or partly clustered, dark brown, spherical, 95 to 130 μm in diameter, and contained 6 to 16 asci. Appendages were mycelioid, numbering 10 to 24 per chasmothecium, 0.5 to 2.5 times as long as the chasmothecial diameter, 1 to 4 septate, and were brown at the base and becoming paler toward the tip. Asci were short stalked, 50 to 75 × 32 to 42 μm and contained two spores. Ascospores were ellipsoid-ovoid with a dimension of 22 to 30 × 15 to 18 μm. On the basis of these morphological characteristics, this fungus was identified as Golovinomyces ambrosiae (Schwein.) U. Braun & R.T.A. Cook (= G. cichoracearum var. latisporus (U. Braun) U. Braun) (1). To confirm the identification, the complete internal transcribed spacer (ITS) region of rDNA from KUS-F24683 was amplified with the primers ITS5 and P3 and sequenced (4). The resulting sequence of 508 bp was deposited in GenBank (Accession No. JF907589) and was identical to the ITS sequences of G. ambropsiae on A. artemisiifolia var. elatior from Japan (AB077631) and Korea (JF919680) as well as on A. trifida from the United States (AF011292). Therefore, the sequence analysis verified the pathogen to be G. ambrosiae. To our knowledge, this is the first record of powdery mildew infections on giant ragweed outside of North America (2). Although the disease incidence is still low, the disease could be a limiting factor to suppress the expansion of this noxious weed in Korea.