Cloning, expression and activity analysis of cutinase from Sclerotinia sclerotiorum / 生物工程学报

Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.

Expression and purification of the transcription factor StMsn2 from Setosphaeria turcica in Escherichia coli

Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.

Contribution of dark septate fungi to the nutrient uptake and growth of rice plants

ABSTRACT The use of dark septate fungi (DSE) to promote plant growth can be beneficial to agriculture, and these organisms are important allies in the search for sustainable agriculture practices. This study investigates the contribution of dark septate fungi to the absorption of nutrients by rice plants and their ensuing growth. Four dark septate fungi isolates that were identified by Internal transcribed spacer phylogeny were inoculated in rice seeds (Cv. Piauí). The resulting root colonization was estimated and the kinetic parameters Vmax and Km were calculated from the nitrate contents of the nutrient solution. The macronutrient levels in the shoots, and the NO3--N, NH4+-N, free amino-N and soluble sugars in the roots, sheathes and leaves were measured. The rice roots were significantly colonized by all of the fungi, but in particular, isolate A103 increased the fresh and dry biomass of the shoots and the number of tillers per plant, amino-N, and soluble sugars as well as the N, P, K, Mg and S contents in comparison with the control treatment. When inoculated with isolates A103 and A101, the plants presented lower Km values, indicating affinity increases for NO3--N absorption. Therefore, the A103 Pleosporales fungus presented the highest potential for the promotion of rice plant growth, increasing the tillering and nutrients uptake, especially N (due to an enhanced affinity for N uptake) and P.

Comparative proteomic analyses of Hyphozyma roseonigra ATCC 20624 in response to sclareol

Abstract Sclareol is an important intermediate for ambroxide synthesis industries. Hyphozyma roseonigra ATCC 20624 was the only reported strain capable of degrading sclareol to the main product of sclareol glycol, which is the precursor of ambroxide. To date, knowledge is lacking about the effects of sclareol on cells and the proteins involved in sclareol metabolism. Comparative proteomic analyses were conducted on the strain H. roseonigra ATCC 20624 by using sclareol or glucose as the sole carbon source. A total of 79 up-regulated protein spots with a >2.0-fold difference in abundance on 2-D gels under sclareol stress conditions were collected for further identification. Seventy spots were successfully identified and finally integrated into 30 proteins. The up-regulated proteins under sclareol stress are involved in carbon metabolism; and nitrogen metabolism; and replication, transcription, and translation processes. Eighteen up-regulated spots were identified as aldehyde dehydrogenases, which indicating that aldehyde dehydrogenases might play an important role in sclareol metabolism. Overall, this study may lay the fundamentals for further cell engineering to improve sclareol glycol production.

Subcutaneous infection by Graphium basitruncatum in a heart transplant patient

ABSTRACT Graphium basitruncatum, a synanamorph of Pseudoallescheria has been rarely reported in human infections. We report a case of subcutaneous phaeohyphomycosis caused by this fungus in a heart transplant recipient. We also describe the phenotypic, molecular methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) used to achieve isolate identification.

Caracterización morfológica y molecular de una colección de aislamientos de Phomopsis longicolla(teleomorfo desconocido: Diaporthales)de la región templada y subtropical de Argentina

Diaporthe(telomorfo) - Phomopsis(anamorfo) (DP) constituye un grupo fúngico de amplia diversidad genética con más de 900 especies distribuidas en un amplio rango de hospedantes que incluye especies cultivadas y no cultivadas, forestales, frutales y malezas. Los aislamientos de DP son hemi-biótrofos y disponen de diferentes fuentes de inóculo primario, como el rastrojo y las semillas, para reiniciar sus ciclos de parasitismo-saprofitismo. Ellos colonizan los tejidos del hospedante desde los estadios tempranos del desarrollo y establecen relaciones nutricionales de endofitia y necrotrofia fúngica. La plasticidad del género Phomopsisha favorecido su expansión a diferentes agro-ecosistemas y hospedantes constituyendo un importante riesgo epidemiológico. El objetivo fue validar la identidad y evaluar las relaciones biológicas de 12 aislamientos de P. longicollay D. phaseolorumvar. sojaeobtenidos en distintos agro-ambientes templados y subtropicales de Argentina, para analizar la variabilidad y estrategias de conservación de la bio-diversidad fúngica. Las cualidades macro-morfo-lógicas(textura y color de colonias, forma y distribución de estromas, desarrollo, forma y distribución de cuerpos fructíferos), y los caracteres micro-morfológicos(tamaño y forma de conidios, ascos y ascosporas) permitieron identificar a tres nuevos aislamientos de P. longicollaincluidos en el complejo D/P. El análisis molecular complementario corrigió las limitaciones derivadas de la caracterización basada sólo en marcadores morfológicos y logró reubicar al aislamiento AFP.8413 de identidad dudosa, en el nodo correspondiente a P. longicolla.De esta manera, la caracterización molecular definió la identidad de los aislamientos y los ubicó en los 4 taxones del complejo DP: diez aislamientos fueron incluidos en Plo(AFP.Gpo 4.4, AFP.Gpo 3.5, AFP.Gpo 4.3, AFP.Gpo 3.4, AFP.CaA, AFP. CaB, AFP.B5L16, AFP.B4L17, AFP.227B2, AFP.8413), un aislamiento incluido en Dps(AFP.Qcol7), un aislamiento en Dpc(AFP.Dpc16) y dos aislamientos incluidos en Dpm(AFP.Dpm109 y AFP.Dpm112). La adecuada identificación de P. longicollay el avance en el conocimiento de las relaciones biológicas (hibridaciones homo o heterotálicas) entre variedades de D. phaseolorum (P. phaseoli)y especies de Diaporthe- Phomopsispermiten comprender la plasticidad para colonizar un amplio rango de hospedantes, los mecanismos de variabilidad genética y la preservación de la diversidad fúngica.

Diaporthe(teleomorpho)-Phomopsis (anamorph) (DP) is a fungal group of great genetic diversity with over 900 species associated to a wide host range that includes cultivated and uncultivated species, forest, fruit trees and weeds. DP isolates are hemi-biotrophs and have different sources of primary inoculum as stubble and seeds to restart cycles of parasitism -saprophytism. They colonize host tissues from early plant stages and establish different nutritional relationships, acting as endophytic and necrotrophic fungi. The plasticity of the Phomopsisgenus has favored its expansion to different agro-ecosystems and various hosts constituting an epidemiological risk. The objective was to validate the identity and evaluate the biological relationships among 12 isolates of P. longicollaand D. phaseolorumvar. sojae(anamorph P. phaseolivar. sojae)obtained in different tempered and subtropical agro-environments of Argentina, in order to analyze the variability and strategies for preserving fungal biodiversity. Macro-morphological attributes (such as texture and color of colonies, stroma shape and distribution, pycnidia and perythecia shape and distribution) and micro-morphological characteristics (such as size and shape of conidia, asci and ascospores) allowed identifying three new isolates as P. longicolla.A complementary molecular analysis was also made to overcome the limitations derived from the morphological analysis, thus the AFP.8413 isolate was finally identified as P. longicolla.The molecular characterization was useful to identify the evaluated isolates and to group them in four taxa of the Diaporthe-Phomopsiscomplex: ten isolates were included in P. longicolla,one isolate was included in D. phaseolorumvar. sojae(anamorph P. phaseolivar. sojae),one isolate was identified as D. phaseolorumvar. caulivoraand two isolates were included in D. phaseolorumvar. meridi-onalis.The use of phenotipic and molecular tools have contributed to an accurate identification of P. longicolla,and comprehension about the biological relationships (homo or heterothallic hibridizations) among D. phaseolo-rumvarieties (P. phaseoli)and species of Diaporthe-Phomopsis.This allowed also a better understanding of the mechanisms of fungic plasticity, to colonize and expand their host range and genetic variability, promoting thus their biodiversity conservation.

Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.

Infection of the lymphatic system by Aureobasidium pullulans in a patient with erythema nodosum leprosum

Aureobasidium pullulans is a causal agent of phaeohyphomycosis, occasionally found in men and animals. As an agent of different opportunistic fungal processes, it may cause fungemia, systemic infections and abscesses in different viscera. This paper aims to report a case of a patient with infection of the lymphatic system by A. pullulans. A 23-year-old patient being treated for erythema nodosum leprosum presented a 60-day complaint of daily fever, hoarseness, odynophagia and weight loss. Laboratory tests showed pancytopenia with severe neutropenia, cervical adenomegaly and solid contrast uptake lesion in the oropharyngeal region. Due to neutropenia and sepsis the patient was initially treated with cefepime and vancomycin, but there was no clinical improvement. Lymph node puncture-aspiration showed yeast-form fungus identified as A. pullulans by sequencing ITS region. The patient was treated with amphotericin B deoxycholate, leading to complete recovery of bone marrow function and regression of adenomegaly and the oropharyngeal lesion.

Subcutaneous Phaeohyphomycosis Caused by Phaeoacremonium Species in a Kidney Transplant Patient: The First Case in Korea / 대한진단검사의학회지

Phaeohyphomycosis is a subcutaneous infection caused by dark pigmented fungi, including fungi of the species Phaeoacremonium, Alternaria, Exophiala, and Pyrenochaeta. In August 2005, a 54-yr-old man who had received a renal transplant 5 yr ago was admitted to our hospital with a subcutaneous mass on the third finger of the right hand; the mass had been present for several months. He had been receiving immunosuppressive agents for several years. He underwent excision of the mass, which was followed by aspiration of the wound for bacterial and fungal cultures. Many fungal hyphae were observed on the histology slide treated with periodic acid-Schiff stain. A few white waxy colonies with a woolly texture grew on the Sabouraud dextrose agar at 30degrees C and changed to dark brown in color. Nucleotide sequencing of internal transcribed spacer regions revealed 100% homology to the Phaeoacremonium aleophilum anamorph and Togninia minima teleomorph (514 bp/514 bp). The patient completely recovered after wide surgical excision. Here, we report the first case of phaeohyphomycosis caused by Phaeoacremonium species in a kidney transplant patient in Korea.

Detección de variabilidad genética en aislamientos de Cercospora kikuchii contaminantes de un mismo sembradío de soja / Detection of genetic variability in Cercospora kikuchii isolates from a single soybean field

El conocimiento de la epidemiología y la estructura poblacional de Cercospora kikuchii está poco desarrollado y no se han comunicado estudios al respecto en la Argentina. El objetivo de este trabajo fue seleccionar oligonucleótidos que permitan detectar variabilidad genética en aislamientos de C. kikuchii obtenidos a partir de soja proveniente de un mismo sembradío, mediante la aplicación de RAPD. Se trabajó con 6 aislamientos de C. kikuchii, 5 de ellos se obtuvieron a partir de trozos de tejido enfermo y el restante provenía de una colección de cultivos. De los 7 oligonucleótidos empleados, 5 resultaron útiles para el estudio poblacional de los aislamientos de C. kikuchii.

Current knowledge about epidemiology and population structure of Cercospora kikuchii is little developed and no studies regarding this subject have been reported in Argentina. The aim of this work was to select primers to study genetic variability in C. kikuchii isolated from the same soybean field using RAPD (Random Amplified Polymorphism DNA). RAPD was applied to the DNA of 5 C. kikuchii, isolated from diseased tissue of the soybean in the same field, another isolate, from a strain collection. Out of seven primers, five of them proved to be useful to study the population of C. kikuchii isolates.

Lethargic crab disease: multidisciplinary evidence supports a mycotic etiology

Although lethargic crab disease (LCD) is causing massive mortalities in populations of the mangrove crab Ucides cordatus of Northeastern Brazil, the identity of its etiological agent was hitherto unknown. In this study we provide robust evidence suggesting that LCD is caused by an anamorph Ascomycota (Fungi). We examined specimens of U. cordatus collected from stocks affected by LCD. Histological and TEM methods detected the presence of hyphae, conidia, and condiophores in several host tissues. Moreover, the abundance of fungal stages is negatively associated with crab health. Finally, DNA was isolated from the fungus and a region of its 18S ribosomal gene was sequenced. Phylogenetic analyses not only confirm the diagnosis of the LCD fungus in crab tissues as an ascomycete, but also suggest a close relationship with members of the subphylum Pezizomycotina.

The use of RAPD to characterize Bipolaris sorokiniana isolates

Bipolaris sorokiniana is a phytopathogenic fungus causing diseases of cereal crops such as common root rot, the leaf spot disease, seedling blight, and black point of the grain. Random-amplified polymorphic DNA (RAPD) assay was used to investigate the genetic diversity of 20 isolates collected from different cultivars in wheat-producing regions in Brazil. Seventy primers, with random nucleotide sequences, were tested. Reproducibility to amplify the genomic DNA of isolates was found for 30 of the 70 primers tested, generating between 1 and 17 fragments ranging from 0.35 to 2.0 kb (average size). The degree of similarity between samples was calculated through simple association and the dendrogram was assessed using the unweighted pair group method with arithmetical average. After the RAPD analyses 19 isolates were closely grouped, having a similarity coefficient of >or= 78%. Isolate I017 showed very low similarity coefficients, ranging between 38 and 46%. The RAPD analyses provided important information as to the degree of genetic variability and the relationship between the isolates investigated, revealing polymorphism and establishing electrophoretic profiles useful to characterize the phytopathogen.

Phylogenetic position of the yeast-like symbiotes of Tagosodes orizicolus (Homoptera: Delphacidae) based on 18S ribosomal DNA partial sequences

Tagosodes orizicolus Muir (Homoptera: Delphacidae), the endemic delphacid species of tropical America carries yeast-like symbiotes (YLS) in the abdominal fat bodies and the ovarial tissues, like other rice planthoppers of Asia. These YLS are obligate symbiotes, which are transmitted transovarially, and maintain a mutualistic relationship with the insect host. This characteristic has made in vitro culture and classification of YLS rather difficult using conventional methods. Nevertheless, microorganisms of similar characteristics have been successfully classified by using molecular taxonomy. In the present work, the YLS of Tagosodes orizicolus (YLSTo) were purified on Percoll gradients, and specific segments of 18S rDNA were amplified by PCR, cloned and sequenced. Sequences were aligned by means of the CLUSTAL V (DNASTAR) program; phylogenetic trees were constructed with the Phylogeny Inference Package (PHYLIP), showing that YLSTo belong to the fungi class Pyrenomycetes, phylum Ascomycota. Similarities between 98% and 100% were observed among YLS of the rice delphacids Tagosodes orizicolus, Nilaparvata lugens, Laodelphax striatellus and Sogatella fur cifera, and between 89.8% and 90.8% when comparing the above to YLS of the aphid Hamiltonaphis styraci. These comparisons revealed that delphacid YLS are a highly conserved monophyletic group within the Pyrenomycetes and are closely related to Hypomyces chrysospermus.

Sex in fungi: lessons of gene regulation

Fungi have been very useful for gene regulation studies. Mating implicates in a series of events influenced by many types of environmental input that are interpreted into regulatory pathways, including signal transduction. Although various aspects of mating and signal transduction in the yeast Saccharomyces cerevisiae have long been characterized, recent findings in filamentous fungi indicate that pheromones and pheromone receptors may be essential for mating partner recognition and also for nucleus recognition in sorting before meiosis. A brief overview on mating-type genes of ascomycete fungi and recent contributions to the understanding of their role in the regulation of multicellularity and sexual dimorphism is presented in this review

Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD)

Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts