Penicillium astrolabium and Penicillium neocrassum, two new species isolated from grapes and their phylogenetic placement in the P. olsonii and P. brevicompactum clade

We describe two new terverticillate Penicillium species isolated from grapes on the basis of phenotypic and phylogenetic differences from known species. The strains were isolated in the course of a study to establish the mycobiota of grapes in Portugal. Penicillium astrolabium is phenotypically similar to P. olsonii but differs from it by two cultural characters, growth rates and the colony reverse color. P. neocrassum is similar to P. brevicompactum but is readily distinguished by sclerotia production. Phylogenetically P. astrolabium and P. neocrassum are placed respectively in the P. olsonii and P. brevicompactum clade. Multilocus analysis confirmed the genetic distinctiveness of both species. The parsimony trees obtained for ITS-lsu rDNA region and two protein coding genes, calmodulin and beta-tubulin, show congruence for all the species in the Olsonii series: P. brevicompactum, P. bialowiezense, P. olsonii, P. astrolabium and P. neocrassum, indicating that these taxa are genetically well isolated.     

A reappraisal of the terverticillate penicillia using biochemical, physiological and morphological features. I. Numerical taxonomy

Three-hundred-and-forty-eight strains representing the major species of terverticillate penicillia, and including representatives of other closely and distantly related species, were included in a numerical taxonomic study. One-hundred characters were derived from morphological features, physiological and biochemical activities and SEM micrographs. Strains were compared by both Gower's coefficient and Pattern difference, and clustered using the average linkage algorithm. Thirty-seven species or species-complex clusters were recovered at approximately 70% similarity; they generally corresponded to existing taxonomic concepts. Several species were shown to contain variants or chemotypes which were often supported by differences in conidial shape and ornamentation. The use of different types of characters enabled a number of new and previously accepted species to be shown to be either variants or deteriorated examples of other species. Variation in properties both between and within species was considered, particularly in relation to strain stability.     

Production of mycophenolic acid by fungi of the genus Penicillium link

Out of 36 strains of fungi of the genus Penicillium, some of which were isolated from ancient permafrost soils, 14 strains synthesized mycophenolic acid (MPA). Maximal (over 500 mg/l) accumulation of MPA in culture liquid was observed in P. brevicompactum strains (VKM F-457, VKM F-477, and VKM F-1150). This was the first study to detect MPA in representatives of the species P. rugulosum; in three strains of this species (VKM FW-665, VKM FW-717, and VKM FW-733), the level of MPA accumulation exceeded 300 mg/l. The time course of the synthesis of MPA by the P. rugulosum strain VKM FW-733 was studied. It was shown that the synthesis of this metabolite was dramatically intensified at the stationary growth phase (ten days).     

Physiological criteria and mycotoxin production as AIDS in identification of common asymmetric penicillia

The taxonomy of the asymmetric (predominantly terverticillate) penicillia is based on morphological differences that leave identification difficult. The application of physiological criteria facilitated the identification of the common asymmetric penicillia investigated. Changes in the placement of some strains of these penicillia made the connection to mycotoxin-producing ability clearer. The classical criterion of conidium color was deemphasized and replaced by the following criteria: (i) growth on nitrite-sucrose agar and (ii) growth and acid (and subsequent base) production on creatine-sucrose agar (containing bromocresol purple). Other criteria used or developed were: (iii) growth on sorbic acid plus benzoic acid agar (50 + 50 ppm, pH 3.8), (iv) growth on an agar containing 1,000 ppm propionic acid (pH 3.8), (v) growth on an agar containing 0.5% acetic acid, (vi) growth at 37 degrees C, (vii) growth rate on an agar containing 0.1% pentachloronitrobenzene, (viii) production of extracellular tricaproinase, and (ix) fasciculation on a medium containing 10 ppm botran (2,6-dichloro-4-nitroanilin). The pattern of extracellular metabolites after thin-layer chromatography was used as a chemotaxonomic criterion. The species investigated, the number of isolates investigated, and the toxins which some of these isolates produce were: Penicillium roqueforti (18) (patulin), P. citrinum (11) (citrinin), P. patulum (9) (patulin and griseofulvin), P. expansum (patulin and citrinin), P. hirsutum (13), P. brevicompactum (19), and P. chrysogenum (12). Widespread species of the P. cyclopium, P. viridicatum, and P. expansum series of Raper and Thom (A Manual of the Penicillia, 1949) were subdivided into four new groups: "P. crustosum pA" (29) (penitrem A), "P. melanochlorum" (29), "P. cyclopium p" (119) (penicillic acid and infrequently penitrem A), and "P. viridicatum o-c" (43) (ochratoxin A and citrinin). "P. viridicatum o-c" was separated from "P. cyclopium p" due to its ability to grow on nitrite as sole nitrogen source. The species and groups investigated were related to the new taxonomic classification of the genus Penicillium according to Pitt.     

Response of liver and kidney adenylate kinase to fasting and refeeding in three strains of mice

The effects of fasting and refeeding on the AK isozymes in liver and kidney were studied in three strains of mice. Our studies showed that changes in total AK activity and AK isozyme patterns were associated with fasting and refeeding. The AK isozyme changes were strain-dependent, differing in kind and degree among the three strains. It was concluded that species, strain and individual isozyme identities should be included in studies defining changes of enzyme activity owing to changes in physiological conditions.     

Divergence of glyceraldehyde-3-phosphate dehydrogenase isozymes in Saccharomyces cerevisiae complex

Although sake yeasts are placed in Saccharomyces cerevisiae, we have been interested in their difference from the other subgroups of the species, and examined their proteins. When SDS-PAGE patterns of their soluble proteins were compared, specific differences between subgroups were found in their 36,000 Da regions. Proteins isolated therefrom were found to be subunits of three isomers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from their N-terminal amino acid sequences and identified with anti-GAPDH serum. Therefore, comparison of zymogram was carried out by a modified method: denatured monomers were observed and the enzyme activity of their oligomers was not considered. SDS-PAGE patterns of all the sake yeasts differed from those of the other strains of S. cerevisiae. Strains of Saccharomyces bayanus showed uniform patterns which are different from the above two groups. Saccharomyces pastorianus strains resembled S. bayanus and were partly similar to S. cerevisiae in their patterns, in agreement with the hypothesis that S. pastorianus is a hybrid between these two species. Patterns of S. paradoxus appeared to be rather similar to those of sake yeasts. Results on the other species of the genus and on the preliminary experiments on PAGE of native isozymes are also described.     

Screening and identification of a Penicillium brevicompactum strain isolated from the fruiting body of Inonotus obliquus and the fermentation production of mycophenolic acid

Mycophenolic acid (MPA) is a fungal metabolite with a variety of biological activities and widely applied in clinical practices. We herein aimed to isolate a new Penicillium brevicompactum strain from the fruiting body of Inonotus obliquus to improve the production of MPA. The fruiting body of Inonotus obliquus was used to isolate P. brevicompactum strains. Identification of the P. brevicompactum strain was performed by sequencing and phylogenetic tree analysis. HPLC assay was conducted to identify the production of MPA. Submerged liquid fermentation and bi-directional fermentation were applied to improve the yield of MPA. The candidates of the P. brevicompactum strain were isolated and screened from the fruiting body of Inonotus obliquus collected from Changbai mountains in China. Based on sequencing and phylogenetic tree analysis of 18S rDNA-ITS, the strain MC-4 was finally identified as P. brevicompactum. And HPLC assay indicated that the isolated P. brevicompactum strain could produce MPA in metabolic products. The optimized conditions of submerged liquid fermentation were as follows: 100 g/L Chinese yam in a liquid PDB medium, pH 6, fermentation temperature of 24 °C, shaker speed of 130 r/min, and fermentation time of 6 days. The maximum value of MPA production was 1.415 g/L after submerged liquid fermentation. Furthermore, the yield of MPA could be significantly increased to 1.537 g/L after bi-directional fermentation with the extractive from fructus Swietenia macrophylla (FSM). We demonstrated that a Penicillium brevicompactum strain isolated from the fruiting body of Inonotus obliquus can be used to improve the production of MPA by submerged liquid fermentation and bi-directional fermentation. This would provide a novel approach for more efficient and safer production of MPA.     

Expression of Pectinase Activity Among Aspergillus Flavus Isolates from Southwestern and Southeastern United States

Aspergillus flavus is a widely distributed filamentous fungus that contaminates crops with the potent carcinogen aflatoxin. This species can be divided into S and L strains on the basis of sclerotial morphology. During crop infection, A. flavus can secrete a large array of hydrolytic enzymes. These include pectinase, which aids fungal spread through plant tissues. A survey of pectinase expression by soil isolates derived from different regions of the United States revealed geographic polymorphisms. Strain L isolates from Arizona produced moderate to high levels of a specific pectinase P2c, while S strain isolates produced variable amounts of P2c. In contrast, L strain isolates from southeastern U.S. yielded variable P2c production, while S strain isolates consistently expressed high P2c levels. These results were corroborated by pectinase surveys of additional collections of A. flavus from soil and cottonseed. Expression patterns for P2c and pectinmethylesterase were evaluated for a select number of isolates using an isoelectric focusing technique. Clear zone reactions from the pectinase plate assay corresponded to the presence of P2c, while red ring reactions corresponded to the lack of P2c. Commercial cottonseed infected by S strain isolates frequently contained aflatoxin, even when infected by S strain isolates that did not produce pectinase P2c. Thus, although P2c-lacking isolates have reduced invasiveness, these isolates still have sufficient pathogenicity to cause aflatoxin contamination.     

Isoelectric focusing studies of aldehyde dehydrogenases from mouse tissues: variant phenotypes of liver, stomach and testis isozymes

Isoelectric focusing techniques (IEF) were used to examine the tissue distribution and genetic variability of aldehyde dehydrogenases (AHDs) from inbred strains of mice. Twelve zones of AHD activity were resolved which were differentially distributed between tissues. Liver extracts exhibited highest activity for most enzymes, with the exception of isozymes found in stomach (AHD-4) and testis (AHD-4 and AHD-6). Genetic variants for AHD-1 (liver mitochondrial isozyme) and AHD-4 (stomach isozyme) were examined from inbred strains and F1 hybrid animals. The results were consistent with dimeric subunit structures (designated as A2 and D2 isozymes respectively). IEF patterns for activity variants of testis-specific AHD-6 were identical, with 3-banded phenotypes being observed. pI values for the AHD forms as well as for aldehyde oxidase and xanthine oxidase isozymes, which stain in the absence of coenzyme, were reported.     

A Study on Isozymes of Peroxidase of 10 Species in Juglans L.

Isozymes of peroxidase of 10 species in Juglans L. were analyzed by using polycrylamide gel electrophoresis, and, as a result, 16 different patterns of isozymes were observed. Polymophism of isozymes patterns appears within species and more significant differences in pattern between species have been found. “Zymogram distance” was measured for each species pair and section pair. The ten species may be divided into 4 groups according to their “zymogram distance” and specific bands, which accords with the classical taxonomy of Juglans L.. Evolutionary relationship among species and rate of evolution for Juglans L. are discussed.     

Structure and function of beta-blucosidases in Dictyostelium discoideum.

There are two isozymes of beta-glucosidase in developing cells of Dictyostelium discoideum. A procedure for screening large numbers of clones for beta-glucosidase activity was utilized to obtain mutations which directly affect the activity. We recovered seven strains which lack both isozymes and four strains with residual activity in which enzymatic and physical properties of both isozymes are altered. Beta-Glucosidase appears to act as a block to selfing in macrocyst formation as shown by the fact that ssite mating type to form macrocyst-like structures. Immunological evidence utilizing antisera prepared against purified beta-glucosidase-1 demonstrates that most of the glycosidases in Dictyostelium discoideum share a common antigenic determinant which appears to be added post-translationally. The two isozymes of beta-glucosidase share common protein subunits but the antigenic determinant is either lacking or masked in beta-glucosidase-2. This may account for some of the enzymatic and physical differences between the two isozymes.     

Electrophoretic protein patterns and enzyme mobilities in anaerobic coryneforms.

The soluble protein patterns and electrophoretic mobilities of malate and succinate dehydrogenases and catalase have been examined in 25 strains of Propionibacterium acnes, P. granulosum, and P. avidum. A distinctive protein pattern for each species was found, and it was possible also to distinguish the serotypes within P. acnes and P. avidum. Strains of P. acnes, P. granulosum, and P. avidum could be differentiated by the mobilities of their malate dehydrogenases. Catalase activity was detected in the soluble fractions of all strains. Catalases from P. acnes and P. avidum strains had the same mobility, whereas that from P. granulosum was slightly slower. Under the conditions used, succinate dehydrogenase activity could be detected, but the patterns were not distinctive.     

In vitro growth of some species of Ascochyta Lib.

Fungi from the genus Ascochyta are generally facultative saprotrophs, which cause diseases in both monocots and dicots. Over 1 000 species belonging to this genus have been identified, 18 of which infect monocot plants from the family Poaceae. This study analyses the effects of temperature and light on the growth of selected fungi which infect monocots (A. agrostidis, A. avenae, A. brachypodii, A. desmazieri, A. digraphidis, A. ducis-aprutii, A. festucae, A. graminea, A. hordei, A. hordei var. americana, A. hordei var. europea, A. hordei var. hordei, A. melicae, A. phleina, A. skagwayensis, A. sorghi, A. stipae, A. zeicola), grown on three types of media; Potato Dextrose Agar (PDA), Coon??s agar (CN) and oatmeal agar (OMA). The fastest growth among the analyzed fungi at low temperatures was found in Ascochyta melicae, while at high temperatures it was A. zeicola. The fastest in vitro growth (average of all fungi) was observed on CN medium at 20°C (3.4 mm/day), while the lowest on OM medium at 5°C (1.0 mm/day). Radial mycelial growth in dark and the light conditions varied. On average, all isolates grew faster in the dark (3.1 mm/day) than in the light (1.9 mm/day). The greatest effect on the production of pycnidia was found for the isolates. Variation in growth and production of pycnidia depended on temperature, medium and lighting for fungi from the genus Ascochyta infecting monocots. Such variation indicates a potential occurrence of these fungi in different environments.     

离子注入选育霉酚酸高产菌株及其发酵条件研究

离子注入技术是20世纪80年代兴起的一种综合诱变技术,其应用于生物工程已取得了丰硕成果,但在霉酚酸产生菌的诱变育种中的应用还未见报道。短密青霉菌(Penicillium brevicompactum)M_51是从土壤中分离得到的MPA产生菌F_663经过紫外线、微波等诱变处理得到的。为获得霉酚酸的高产工业菌株,进一步对该菌株进行了离子注入诱变处理。用15keV氮离子分5个剂量进行处理,结果显示,随离子注入剂量增加,存活率呈现较明显的下降_上升_下降的“马鞍型”变化趋势。在剂量为140×2.6×1013ions/cm2时,菌株变异率及正变率均最高,分别达到88.9%和63.4%。用HPLC定量测定发酵液中霉酚酸的含量,筛选到产霉酚酸能力提高30.1%的突变株M_163。经过连续传代试验,其遗传性状稳定。对发酵条件的优化结果显示最佳种龄为24h;用正交试验方法对发酵培养基中的碳、氮源进行优化,得到较优配方。突变株M_163在最优发酵条件下,霉酚酸摇瓶发酵单位可达2819μg/mL。野生菌株F_663的MPA产量为133μg/mL,经过5代诱变育种及发酵条件优化,产量提高了20.2倍。     

An electrophoretic analysis of the isozymes of malate dehydrogenase in several different plants

The particulate and soluble fractions of cell-free extracts from seeds, roots, and leaves of 10 different plants were examined electrophoretically for isozymes of malate dehydrogenase. Distinct isozyme patterns were observed for each plant and even for the individual tissues of each species. There were some isozymes in several different plant extracts with equal electrophoretic mobilities, but there was no isozyme band that was common to all tissues or to all plants.     

Alterations in peroxidase activity and peroxidase isozymes in virus-infected plants

Peroxidase activity and isozyme patterns were investigated in two leguminous species infected with viruses which produced either local necrotic or systemic chlorotic symptoms. Highest peroxidase activity was recorded when the hosts reacted to infection with necrotic local lesions. No new virus-specific isozymes were found as a result of infection, but some isozymes, apparently associated with senescence, appeared earlier in extracts from leaves showing necrosis than in extracts from healthy leaves, or from infected leaves showing only very mild chlorosis. Increase in peroxidase activity was accompanied by alteration in isozyme pattern.     

Genetic variation of trypsin and chymotrypsin inhibitors in pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives

Variation in the trypsin inhibitors (TIs) and the chymotrypsin inhibitors (CIs) among 69 pigeonpea [Cajanus cajan (L.) Millsp.] strains from a wide geographical distribution and among 17 accessions representing seven wild Cajanus species was studied by electrophoretic banding pattern comparisons and by spectrophotometric activity assays. The TI and CI electrophoretic migration patterns among the pigeonpea strains were highly uniform but varied in the inhibitor band intensities. The migration patterns of the inhibitors in the wild Cajanus species were highly species specific. The mean TI activity of pigeonpea strains (2279 units) was significantly higher than that of the wild Cajanus species (1407 units). However, the mean CI activity in the pigeonpea strains (62 units) was much lower than that in the wild species (162 units). Kenya 2 and ICP 9151 were the lowest and the highest, respectively, in both the TI and CI activities among all the pigeonpea strains used in this study. A highly-significant positive correlation was observed between the TI and CI activities. The Bowman-Birk type inhibitors with both TI and CI activities were identified in all the pigeonpea strains and also in the accessions of all the wild species except C. volubilis (Blanco) Blanco. The C. volubilis accession ICPW 169 was found to be null for both CI bands and CI activity. Environment, strain, and environment x strain interaction showed highly-significant effects on both the TI and CI activities. Growing the pigeonpea strains at a different environment from their area of adaptation increased TI and CI activities and also altered the maturity period.     

Use of gel electrophoresis for the study of enzymatic activities of cold-adapted bacteria

Glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) activity of 13 cold-adapted strains, isolated from cold soils and showing GDH and/or LDH activity in spectrophotometric assays, were revealed by the use of electrophoresis on a nondenaturing acrylamide gel (zymogram). Psychrophilic strains were grown at 4 degrees C and 10 degrees C and the psychrotolerant strains at 4 degrees, 20 degrees and 28 degrees C. Incubation with the specific substrate and staining were done at 4, 28 or 37 degrees C. In the most cold-adapted strains, LDH and GDH production was high at 4 degrees C. In psychrotrophic strains, enzyme production and activity were greater at 20 or 28 degrees C than at lower temperatures. LDH remained active up to 37 degrees C while GDH activity was more thermolabile. GDH activity was NAD-dependent in some psychrophilic strains. In other strains, it was dependent on NAD(P) only or on both NAD and NAD(P). Two bands were seen for GDH or LDH activity in some strains. This method, which does not require a dialysis step, can be used to study the influence of temperature on enzyme production and activity, and the co-factor dependence. It detects phenotypic differences between isozymes, providing data for systematics.     

Genomic diversity within the genus Pediococcus as revealed by randomly amplified polymorphic DNA PCR and pulsed-field gel electrophoresis

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.     

Molecular characterization and evaluation of pectinase and cellulase production of Penicillium spp.

Penicillium species were analyzed with molecular markers and for pectinase and cellulase production. RAPD and PCR-RFLP analysis indicated high polymorphism among at least 5 of 10 Penicillium species. Five species were chosen for pectinase and cellulase production in liquid medium and four of which appeared similar based on molecular analyses. P. brevicompactum and P. griseoroseum gave the highest pectinase production and were highly divergent by molecular techniques.