Characterization of Soil Actinomycetes From Malino Pine Forest Rhizosphere of South Sulawesi

Actinomycetes are a group of Gram-positive bacteria that produce active compounds with a wide distribution range in nature especially in soil. The purpose of this study was to isolate actinomycetes from the rhizosphere soil of the Malino pine forest, South Sulawesi. This research is a descriptive qualitative study of 15 actinomycetes isolated from the rhizosphere soil of the Pine Forest. These 15 isolates have been successfully identified to the genus level. Actinomycetes were isolated by direct dilution method and further morphological identification was carried out. Actinomycetes were isolated on yeast malt agar (YMA) medium. The growing isolates showed colony colors brown, gray, and white. Produces yellow, brown, and beige color pigments. Based on the characterization carried out, all isolates were identified into the genus Streptomyces.Abstrak: Aktinomisetes merupakan kelompok bakteri gram positif yang menghasilkan senyawa aktif dengan rentang distribusi yang luas di alam, terutama di tanah. Tujuan penelitian ini untuk mengisolasi aktinomisetes dari tanah rizosfer hutan pinus Malino Sulawesi Selatan. Penelitian ini merupakan penelitian kualitatif deskiptif terhadap 15 isolat aktinomisetes yang diisolasi dari tanah rizosfer Hutan Pinus Malino Sulawesi Selatan. 15 isolat ini telah berhasil didentifikasi sampai tingkat genus. Aktinomisetes diisolasi dengan metode pengenceran secara langsung dan dilakukan identifikasi morfologi lebih lanjut. Aktinomisetes diisolasi pada media yeast malt agar (YMA). Isolat yang tumbuh memperlihatkan warna koloni coklat, abu-abu dan putih. Menghasilkan pigmen warna kuning, coklat dan krem. Berdasarkan karakterisasi yang dilakukan keseluruhan isolat teridentifikasi ke dalam genus Streptomyces.

STABILITY OF THE STRAINS OF BASIDIOMYCETES DURING STORAGE IN THE COLLECTION

Basidial macromycetes may be a material for the development of new biotechnologies, medical preparations, components of dietary nutrition. Therefore, it is necessary for the highest level of quality for maintenance and identification of mushroom strains in the collection. An important parameter, in this case, is a stability of isolated and described collection strains of basidiomycetes. Stability is one of the key issues of long-term preservation of pure culture collections. For the collection of medicinal basidiomycetes of ONU I.I. Mechnikov, which preserves by the method of periodic reseeding of colonies the strain stability had not been studied yet. The goal of this research is to study the stability of this collection by a growth rate of mushroom colonies and electrophoretic spectra of carboxylesterases after different times of storage of cultures on malt agar. In this research the strains of three age categories (1, 2 and 3 years) of storage on malt-agar medium at temperature 4 ° С for were tested. The radial growth rate of their vegetative mycelium and the spectra of multiple molecular forms of carboxylesterases by the method of vertical electrophoresis in 7% of polyacrylamide gel were investigated. It was established that the stability of the radial growth rate of A. auricula-judae, F. velutipes, G. lucidum the vegetative mycelium after different storage periods is high according to the values of variation coefficients. At the same time, the expression of molecular forms of carboxylesterase showed sufficient variability. Partially conservative molecular forms were detected in some age groups of strains, as well as for individual strains. Thus the growth rate of colonies is a stable indicator and the molecular forms of carboxylesterases of different ages strains are variable.

Isolasidan Karakterisasi Morfologi Aktinomiset Indigenus Asal Tanah Gambut

Aktinomiset merupakan kelompok mikroorganisme yang tersebar luas di lingkungan darat, air tawar dan laut. Aktinomiset memainkan peran penting dalam dekomposisi bahan organik dan dengan demikian mengisi pasokan nutrisi dalam tanah.Penelitian ini bertujuan untuk mengisolasi dan karakterisasi morfologi aktinomiset indigenus asal tanah gambut. Isolasi aktinomiset dari contoh tanah gambut yang memiliki karakteristik pH 4.7, rasio C/N 38 dan nilai KTK 70.5 asal Tanjung Jabung Barat, Jambi.Isolat yang diperoleh dimurnikan dan diremajakan kembali pada media Yeast Malt Agar (YMA). Isolat aktinomiset yang telah murni ditumbuhkan pada tiga jenis media pertumbuhan yaitu Yeast Malt Agar (YMA), Yeast Starch Agar (YSA), dan Oatmeal Agar (OM) selama ±10 hari pada suhu ruang. Selanjutnya, dilakukan pengelompokan isolat aktinomiset berdasarkan warna koloninya. Terdapat 80 koloni aktinomiset yang berhasil diisolasi dari contoh tanah gambut, yang tumbuh pada media agar-agar HV dan dimurnikan di media YMA. Hasil seleksi berdasarkan keragaman bentuk koloni dari 80 isolat diperoleh 20 isolat murni yang memiliki beragam morfologi koloni, menghasilkan miselia aerial dan mampu tumbuh baik serta bersporulasi pada umur 7-14 hari. Karakteristik morfologi ini mengindikasikan isolat-isolat tersebut adalah Streptomyces sp.

Ultraestrutura celular e expressão de proteínas de leveduras hanseniaspora sob efeito do estresse etanólico

Resumo O objetivo deste estudo foi avaliar a resposta de Hanseniaspora opuntiae (Ho41) e H. guilliermondii (Hg43) ao estresse etanólico, observando a ultraestrutura e o perfil de expressão proteica em concentrações crescentes de etanol. A ultraestrutura foi analisada por microscopia eletrônica de varredura (MEV) e a expressão proteica, pelo perfil eletroforético (SDS-PAGE). Na análise microscópica, as cepas em meio Yeast Malt Agar sem etanol mostraram células jovens com morfologia apiculada, brotamento bilateral e polos distais côncavos. Com o início do estresse, a 3% de etanol, as células apresentaram múltiplas cicatrizes em forma de anéis e, com 6%, alterações na integridade da parede celular, plasmólise e ativação da autólise. Na análise eletroforética, observou-se, tanto para Ho41 quanto para Hg43, aumento na expressão de um peptídeo de 100 kDa, com aumento do etanol no meio, indicando ser uma proteína de choque térmico (HSP). As HSPs vêm sendo patenteadas como marcadores de organismos de interesse biotecnológico, já que as condições necessárias para obtenção de bioprodutos muitas vezes requerem cultivo sob estresse. Neste contexto, esta proteína pode ser indicada como marcador molecular para bioprospecção ou melhoramento genético de cepas não-saccharomyces mais resistentes aos processos de fermentação, na fabricação de vinhos.

Laboratory invesitgations of variability of Ascochyta fabae Speg. isolates obtained from horse bean

Fifty five isolates of <i>Ascochyta fabae</i> Speg. were investigated. They were selected from 1650 isolates of this fungus obtained from horse bean seeds in 1974-1976. All the isolates grew and sporulated on Potato Dextrose Agar, Malt Agar and Horse Bean Agar. The rate of their growth amounted to 1-4 mm per 24 hours. The variability of isolates in size of pycnidia and conidia and number of sepia was noticed. A few spores with untypical shapes were observed as well.

First Report of Umbel Browning and Stem Necrosis Caused by Diaporthe angelicae on Carrot in France

In 2011, carrot (Daucus carota L.) seed production occurred on 2,900 ha, which accounts for approximately 25% of the area devoted to the production of vegetable fine seeds. Since 2007, symptoms of umbel browning have been regularly observed in carrot production areas located in the central region. Initially, triangular necrotic lesions appeared on carrot umbels that later spread to the entire umbels and often progressed to the stems. Diseased umbels became dried prematurely, compromising seed development. The loss in seed production was estimated at approximately 8% of the harvested carrot umbels during the cropping seasons of spring and summer 2007 and 2008 in France. In collaboration with seed companies, diseased carrot stems were collected from seven fields of seed production (eight plants per field) and a fungus was isolated from the tissue. The cultures were grown on malt (2%) agar (1.5%) medium and incubated for 2 weeks at 22°C in darkness. Young fungal colonies were white and a brownish green pigmentation developed when the colonies became older. The same color was observed from the top and on the reverse of the colonies. To induce sporulation, isolates were grown on water agar (1.5%) medium in the presence of carrot stem fragments for 1 week at 22°C in darkness, followed by 1 week at 22°C in white light under a 16-h photoperiod. Pycnidia were produced on stem fragments and contained alpha and beta conidia typical of the genus Diaporthe (2). Alternatively, pycnidia were also obtained on malt agar medium after 2 weeks of culture at 25°C in white light under a 12-h photoperiod. The size of alpha and beta conidia was 6.3 ± 0.5 × 2.3 ± 0.4 μm and 23.3 ± 1.8 × 0.9 ± 0.2 μm, respectively (n = 170). In order to confirm the identification at the genus level and determine the species, DNA was extracted from the mycelium of three representative isolates and the ITS regions of the ribosomal DNA were amplified using universal primers (1). The sequences of the amplified products (GenBank Accession Nos. KF240772 to KF240774) were 100% identical with the ITS sequence of a Diaporthe angelicae isolate deposited in the NCBI database (CBS 111592 isolate, KC343027). To confirm pathogenicity, the three isolates of D. angelicae were inoculated on carrot umbels in the greenhouse. A total of nine plants were inoculated (three plants per isolate). Using a micropipette, 10 μl of a conidial suspension containing alpha and beta conidia (105 conidia mL–1) were deposited at the base of the primary umbel and two secondary umbels, which were wounded before inoculation using a scalpel blade. Seven inoculated plants developed triangular, necrotic lesions that were typical umbel browning. D. angelicae was re-isolated on malt agar medium from the inoculated diseased carrot umbels. To our knowledge, this is the first report of D. angelicae in carrot cultivated for seed production in France. The disease resembles the lesions described in the Netherlands in 1951 on carrot inflorescence caused by Phomopsis dauci (3). In future experiments, it would be crucial to precisely determine if D. angelicae could be transmitted to the seeds. References: (1) M. A. Innis et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (2) J. M. Santos and A. J. L. Philips. Fungal Divers. 34:111, 2009. (3) J. A. von Arx. Eur. J. Plant Pathol. 57:44, 1951.

Mycobiota of rape seeds in Romania. I. Identification of mycobiota associated with rape seeds from different areas of Romania

<p>The spectrum of fungal diversity associated with rape seeds belonging to 33 cultivars (Alaska, Astrada, Astrid, Atlantic, Betty, Champlein, Chayenne, Dexter, Digger, Elvis, Eurowest, Finesse, Herkules, Hydromel, Hydromel-MA, Ladoga, anitoba, Masa Rom, Milena, Mohican, Montego, Nectar, Ontario, Orkan, Perla (4 lots), Remy, Robust, Rodeo, Saphir, Tiger, Tiger CBC Lot ROM06-121-110, Triangle, Valesca, Vectra) and 2 hybrids (H-90-20-83, H-90-21-83) has been established by samples’ macroscopical and microscopical analizying, during 2006-2008, for the first time in Romania. The Ulster method on malt-agar and PDA culture media has been used, evaluating the percentage of fungal taxons present on/in rape seeds. The most important pathogenic fungi identified were: <em>Sclerotinia sclerotiorum </em>(Lib.) de Bary, <em>Botrytis cinerea </em>Pers., <em>Rhizoctonia solani </em>Kühn, <em>Alternaria brassicae </em>(Berk.) Sacc., <em>A. brassicicola </em>(Schwein.) Wiltshire and <em>Fusarium </em>spp. Also, a large quantities of some saprophytic fungi, as <em>Alternaria</em>, <em>Cladosporium</em>, <em>Aspergillus</em>, <em>Penicillium</em>, <em>Rhizopus </em>have been recorded. These ones have been affected the health condition of rape seeds, suppressing their germination and other vital phenomena. Among potential antagonistic fungi the following genera have been isolated: <em>Chaetomium </em>(0-4%), <em>Trichoderma </em>(0-10%), <em>Aspergillus </em>(0-14%), <em>Penicillium </em>(0-100%). Some correlations and comparisons have been established between fungal diversity, their provenience, cultivars, culture media (Malt-Agar/MA, Potato-Dextrose-Agar/PDA) used. It has been evaluated the behaviour of rape cultivars and hybrids towards the main rape seed pathogens.</p>

Fungi colonizing various organs of thyme Thymus vulgaris L. cultivated in the region of Lublin

In 1998-2001, the healthiness of thyme cultivated in the region of Lublin was examined. Surveys were made on the one-year-old plantations of thyme at a stage of 6-week-old seedlings and just before the first harvest of the crop, as well as on the two-year-old plantations in spring and before the last harvest. The percentage of the plants showing fungal disease symptoms and the index of infection with fungi were determined. The fungi were isolated from superficially disinfected plant fragments namely from roots, bases of stem and leaves, separately, using mineral culture medium. PDA and SNA media were used to culture <i>Fusarium</i> spp., malt-agar and Czapek-Dox ones to culture <i>Penicillium</i> spp. and malt-agar, oat-agar and cherry-agar ones to culture <i>Phoma</i> spp. The percentage of plant infected with the fungi ranged within 12.18 and 23.05, in case of the one-year-old plantations, and within 29.91 and 43.65 in the two-year-old ones, whereas values of the index of infection ranged within 11.56 and 24.69 and within 20.75 and 43,28, respectively. Necroses were observed on roots and base of stems on one-year-old and two-year-old plantations, but in the last period of vegetation of thyme close to harvest. very often stems and leaves showed symptoms of a complete necrosis. It was found that base of stems and roots of thyme in the first and the second year of cultivation were colonized by a complex of pathogenic fungi:<i>Fusarium spp., Rhizoctonia solani, Thielaviopsis basicola</i> were obtained from the major part of diseased plants. Among the <i>Fusarium</i> species colonizing bases of stems <i>F.culmorum, F.avenaceum, F.equiseti</i> and <i>F.oxysporum</i> dominated, but from roots of thyme most often <i>F.oxysporum, F.equiseti and F.culmorum</i> were isolated. From stems, and particularly from leaves of thyme showing dark spots, commonly <i>Alternaria alternata</i> was obtained. On the other hand, shoots and leaves, but rarely roots of thyme, were colonized by various species of <i>Phoma</i>, particularly by <i>Phoma exigua</i> var. <i>exigua. Colletotrichum gleosporioides</i> occurred rarely on thyme in the area surveyed.

Fungi transporting by sowing seed material of herbs

Sowing seed material of33 species of herbs obtained in 1997-1999 from the Herb Seed-Testing Station, in Bydgoszcz were examined. Fungi were isolated using the method of artificial cultures on the mineral medium. Sixty seeds superficially disinfected and sixty undisinfected seeds were taken from each sample. Obtained single-spore cultures of the fungi grown on malt-agar or on standard medium were identified up to the species level. Fungi species belonging to the genus <i>Fusarium</i> were identified on the PDA and SNA, <i>Aspergillus</i> spp. and <i>Penicillium</i> spp. on the malt-agar and Czapek-Dox and <i>Phoma</i> spp. on the malt-agar, oat-meal-agar and cherry-agar. Mycological analyses showed that the superficial disinfection of seeds reduced by three times the number of isolates obtained. The fungi most frequently isolated from both the inside and the outside seed tissues were <i>Botrytis cinerea, Phoma exigua</i> var. <i>exigua</i> and species of <i>Alternaria, Epicoccum, Fusarium, Penicillium, Phyllosticta, Rhizopus, Trichothecium</i>.

The studies on ash dying (Fraxinus excelsior L.) in the Włoszczowa Forest Unit stands

The studies were carried out in the Włoszczowa Forest Unit, in 9 ash stands differing in respect of age, origin (natural, artificial), site and in the nursery on 3 quarters differing due to a silvicultural method (transplanted and not transplanted) and seedlings age. In each stand an analysis of disease symptoms was carried out on 100 trees (2 - 20 years old stands) or 50 trees (21 - 80 years old stands) growing side by side in central part of the stand, while in the nursery in each block 200 seedlings were analyzed (4 sectors with 50 seedlings each). From the infected seedlings and trees 120 fragments of dead branches, living branches with cankers, and dead roots were taken. Identification of fungi was made on the basis of fructification and over 300 isolations of fungi on malt agar medium. The most frequent disease symptoms in ash stands were: the dead top (34.7% trees), the dying of whole branches (83.5%), the dying of the top of branches (20.1%), the occurrence of healed (36.0%) and unhealed cankers (18.9%) and the slime flux (23.7%) on the trunk, also the chlorosis of leaves (7.5%) and their atrophy (11.2%). Most of the types of disease symptoms appeared irrespectively of the tree age, origin and site, sometimes showing only a difference in the frequency of occurrence. On the seedlings in the nursery the shoot discolouration, healed and unhealed cankers on shoots and necrosis of a part of leaves were recorded most frequently. Disease symptoms occurred more frequently on 4-year-old seedlings in comparison with 3-year-old. In respect of transplanted seedlings the leaves dying was more frequent. Within cankers and on dead tops of shoots the most frequent were: <i>Alternaria alternata</i>, <i>Chalara</i> sp., <i>Cytospora ambiens</i>, <i>Diplodia mutila</i>, <i>Fusarium lateritium</i>, <i>Gloeosporidiella turgida</i>, <i>Phomopsis controversa</i> and <i>Phomopsis scobina</i>. In sparsely found dead roots of living trees appeared mostly: <i>Cryptosporiopsis radicicola</i>, <i>Cylindrocarpon destructans</i> and <i>Phialocephala</i> sp.