A re-examination of the phylogenetic relationship between the causal agents of carrot black rot, Alternaria radicina and A. carotiincultae

The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined based on morphology, sequence analysis of rDNA (ITS and mitochondrial small subunit [mtSSU]), protein coding genes (actin [ACT], beta-tubulin, chitin synthase [CHS], translation elongation factor [EF-1a], Alternaria allergen a1 [Alt a1], and glyceraldehyde-3-phosphate dehydrogenase [gpd]), and RAPD and ISSR analysis of total genomic DNA. Although some morphological characters overlapped to a degree, with A. radicina isolates expressing moderate variation and A. carotiincultae isolates being highly uniform, A. carotiincultae could be differentiated from A. radicina based on significantly greater growth rate on potato dextrose agar (PDA) or acidified PDA (APDA) and average number of transverse septa per conidium. Sequence of rDNA and two protein coding genes, ACT and CHS, were invariant between species. However polymorphism with the EF-1a, beta-tubulin, and Alt a1 gene strictly separated the population of A. radicina and A. carotiincultae as distinct lineages, as did RAPD and ISSR analysis. The polymorphic gpd gene did not strictly separate the two species. However isolates of A. radicina encompassed several haplotypes, one of which was the exclusive haplotype possessed by A. carotiincultae isolates, suggesting evidence of incomplete lineage sorting. The results suggest that A. carotiincultae is closely related to A. radicina but is a recently divergent and distinct lineage, which supports its status as a separate species.     

Brefeldin A,a cytotoxin produced by Paecilomyces sp. and Aspergillus clavatus isolated from Taxus mairei and Torreya grandis

Paecilomyces sp. and Aspergillus clavatus, which were isolated from Taxus mairei and Torreya grandis from southeast China, produced toxic metabolites when grown in liquid culture. Nuclear magnetic resonance techniques, infrared spectrometry, electrospray ionization mass spectroscopy and X-ray analysis identified brefeldin A, a bioactive metabolite produced by a number of fungal species belonging to the genera Alternaria, Ascochyta, Penicillium, Curvularia, Cercospora and Phyllosticta. This is the first report of the isolation of the cytotoxin from Paecilomyces sp. and A. clavatus. The relevance of brefeldin A to the association between these fungi and their host plants is discussed.     

Antifungal activity of venenatine, an indole alkaloid isolated fromAlstonia venenata

The indole alkaloid venenatine exhibited antifungal activity against some plant pathogenic and saprophytic fungi. Venenatine in an aqueous acetic acid solution inhibited spore germination of all the 10 tested fungi, Fusarium udum, Alternaria brassicicola, Ustilago cynodontis and Aspergillus flavus showed an especially high sensitivity towards this compound, exhibiting germination levels below 10%. The spore germination and colony development of the parasitic fungus Erysiphe pisi, which causes powdery mildew in pea (Pisum sativum), on excised leaves of pea was also significantly affected. Pre-inoculation rather than post inoculation treatment of the leaves was more inhibitory against spore germination and colony development.     

Spore production and mycorrhizal development in various tropical crop hosts infected with Glomus clarum

Plant growth, mycorrhizal development and vesicular arbuscular spore production were examined in five tropical crop host species inoculated with Glomus clarum and grown in a glasshouse. In one of the two experiments, sequential harvests of maize, sorghum and chickpea were made in order to study spore production in relation to plant growth and mycorrhizal development. Spore numbers in each of these hosts increased at a fairly constant rate until maximum plant dry weight, when spore production ceased. Sorghum and maize produced considerably more spores than chickpea, with spore numbers being closely correlated with mycorrhizal root length. In the second experiment, Glomus clarum was cultured on each of maize, millet, sorghum, groundnut and chickpea for three consecutive generations before cross-inoculation of the spores from each host onto all five hosts. Sporulation with respect to host size was generally greatest when the inoculum used to infect a host had been produced on that host. The growth-promoting effects of the fungus were not influenced by the source of the inoculum. More spores were produced on the cereals than the legumes. Differences in spore numbers amongst hosts and plant generations were apparently influenced mainly by infected root length and by the growth period.     

Phytotoxins from Alternaria helianthi: radicinin,and the structures of deoxyradicinol and radianthin

A novel compound, radianthin, with phytotoxic activity was isolated from liquid cultures of Alternaria helianthi and identified as a pyrone related to radicinin. A second metabolite was identified as radicinin itself while deoxyradicinol is described for the first time as a natural product.     

Community of arbuscular mycorrhizal fungi in a coastal vegetation on Okinawa island and effect of the isolated fungi on growth of sorghum under salt-treated conditions

Community of arbuscular mycorrhizal (AM) fungi in a coastal vegetation on Okinawa island in Japan was examined. A sampling plot was established in a colony of Ipomoea pes-caprae (Convolvulaceae) on the beach in Tamagusuku, Okinawa Pref, in which eight root samples of I. pes-caprae and three root samples each of Vigna marina (Leguminosae) and Paspalum distichum (Poaceae) were collected. Partial 18S rDNA of AM fungi was amplified from the root samples by polymerase chain reaction (PCR) with primers NS31 and AM1. Restriction fragment length polymorphism analysis with HinfI and RsaI for cloned PCR products revealed that two types of Glomus sp., type A and type B, were dominant in the colony. Among them, the fungi of type A were especially dominant near the edge of the colony facing the sea. A phylogenetic analysis showed that the AM fungi of type B are closely related to Glomus intraradices and those of type A are nearly related to type B. From the sequence data, it was also found that type A was further divided into two types, type A1 and A2. One representative strain each of the three types, type A1, A2, and B, propagated from single spore each, was examined for the growth of sorghum (Sorghum bicolor) at three different salinity levels, 0, 100, and 200 mM NaCl. At the non-salt-treated condition, the type B fungus was the most effective on shoot growth enhancement of the host plant, whereas at the salt-treated conditions, the type A2 fungus was the most effective. An efficient suppression of Na + translocation into the shoot by the examined AM fungi was found. These results suggested that the AM fungi dominant near the sea are adapted to salt-stressed environment to alleviate the salt stress of host plants.     

Melanin production in Alternaria helianthi

Abstract

Most of the plant pathogenic fungi produce a dark phenolic polymer called melanin. The high performance liquid chromatography (HPLC) analysis of the mycelial extract of Alternaria helianthi revealed an accumulation of scytalone and a shunt metabolite 2-hydroxyjuglone which confirms the production of dihydroxynapthalene type of melanin. The growth and melanin of A. helianthi increased when grown in host extract broth at 6.5 pH and a temperature beyond 30°C had an inhibitory effect on the pathogen. The production and type of melanin produced in Alternaria helianthi is reported for the first time.     

The effect of alkanes on the physiology and metabolism of the entomopathogenic fungus,Isaria fumosorosea

The influence of different alkanes on spore morphology, glyoxlate pathway enzyme activities, total lipid contents and fatty acid composition of Isaria fumosorosea were investigated under laboratory conditions. Fungal spores grown on different alkanes showed higher germination and mycelial growth when compared to control. A strong induction of glyoxlate cycle enzymes in cell-free extracts was observed for cells grown on different alkanes when compared to glucose and control. Higher activities of glyoxlate cycle enzymes were observed for cells grown on alkanes when compared to other treatments. Even numbered fatty acids accounted for the majority of fatty acid production with a significant increase in relative amounts of linoleic acid and palmatic acid observed for conidia grown on alkanes. These results indicate that addition of alkanes to culture media can be a tool to pre-induce metabolic adaptations that can facilitate successful infection of insect host by entomopathogenic fungi.     

基于rDNAITS序列分析Alternaria与相似属的关系及A.leucanthemi的分类地位

对Alternaria,Stemphylium,Ulocladium,Nimbya,Embellisia等五个形态相似属的代表性种进行了5.8S/ITS区段序列测定,连同从GenBank下载的相关有性型Pleospora,Lewia的同项资料,由Neighbor-joining方法构建系统发育树。序列对比结果显示,所有参试的菌株/种的5.8SrDNA序列保守程度较高,ITS序列变化较大,ITS1比ITS2变化更大。由5.8S/ITS构建的系统发育树可以足够将供试菌区分到属;系统发育树显示Alternaria,Ulocladium,Nimbya和Embellisia系统学关系较近,又存在一定的分化;Stemphylium聚在相对较远的分支上,与上述四属关系较远,相对独立进化;有性型的Pleospora与无性型的Stemphylium被聚到一个分支,这种不同阶段菌株在遗传基础上的一致,充分证明所用方法及所选测序的rDNA区段对本类群真菌的分类很有价值。Alternarialeucanthemi位于系统发育树的外侧,较为独立进化,与Alternaria的其它种亲缘关系较远,却与Stemphyliumspp.和Bipolarisspp.关系较近,对其目前的分类地位提出质疑。     

Descriptions and phylogenetic systematics of Myxobolus spp. from cyprinids in Algonquin Park, Ontario

Eight species of Myxobolus were collected from four species of cyprinids in Algonquin Park, Ontario. On the basis of spore morphology, five of these species are described as new and two are redescribed. The evolutionary relationships among these eight species were studied using partial small subunit ribosomal DNA (ssu-rDNA) sequence data. The resulting cladograms, which were highly resolved and with strongly supported relationships, allowed for the evaluation of spore morphology, host specificity, and tissue tropism, criteria traditionally used in species identification. These criteria, recently criticized for creating artificial rather than natural taxonomic groupings, were evaluated for their reliability in the systematics of the species examined. The data showed that distantly related species often infect the same host and tissue, and that closely related species often occur in different hosts. Morphologically similar species are more closely related to each other and the taxonomy based on spore morphology is consistent with the relationships depicted in the phylogenies. These results suggest that spore morphology is better than host specificity and tissue tropism as a species character, as well as for determining evolutionary relationships among the species of Myxobolus examined.     

1‐Phenyl‐3‐pentanone,a Volatile Compound from the Edible Mushroom Mycoleptodonoides aitchisonii Active Against Some Phytopathogenic Fungi

Volatiles produced by mycelia of mushrooms with aromatic odour were investigated for their antifungal activity against plant‐pathogenic fungi. The results of the screening of 23 species of basidiomycetes revealed that volatile substances from mycelia of Mycoleptodonoides aitchisonii (TUFC10099), an edible mushroom, strongly inhibited the mycelial growth, spore germination and lesion formation on host leaves of some plant‐pathogenic fungi including Alternaria alternata, A. brassicicola, A. brassicae, Colletotrichum orbiculare and Corynespora cassiicola. The volatile compounds were isolated from the culture filtrate of M. aitchisonii, and 1‐phenyl‐3‐pentanone was identified as a major antifungal volatile. The compound had significantly inhibitory activity against plant‐pathogenic fungi at 35 ppm. This is the first report that the volatile compound produced by mycelia of M. aitchisonii has antifungal activity against plant‐pathogenic fungi.     

Entomophthora species with E. muscae-like primary spores on two new insect orders, Coleoptera and Hymenoptera

Adults of Cantharis livlda (Coleoptera: Cantharidae) and of Torymus druparum (Hymenoptera: Torymidae) were found naturally infected by fungi from the Entomophthorales in Denmark. The morphology of the primary spores of the two fungi clearly showed that they belong to the genus Entomophthora s.str. No species from this genus has been reported so far from these insect orders. With respect to spore size and number of nuclei per spore, the fungi fall within the range of species from the E. muscae complex, known only from Diptera. A transfer of the fungus from T. druparum to Psila rosae (Diptera: Psilidae) was however possible. The findings thus confirm a significant widening of the host range of species within the E. muscae complex.     

Interactions between water-stress and different mycorrhizal inocula on plant growth and mycorrhizal development in maize and sorghum

Maize (Zea mays) and sorghum (Sorghum bicolor) were inoculated with a range of VAM fungi and grown under water-stressed and unstressed conditions. There was considerable variation amongst the inocula in their effects on plant growth. Inoculation with Glomus clarum produced the biggest plants in each host, with Glomus monosporum and Acaulospora sp. giving the least growth overall. Root infection produced by the different inocula also varied, but levels were not correlated with effects on plant growth. Water-stress reduced plant growth, with the effects not being altered by mycorrhizal infection. VAM infection levels were not affected by water-stress. Spore production from most inocula was reduced by water-stress, both in total spore numbers and in terms of spores per gram plant weight. Sporulation of G. clarum, G. epigeum and G. monosporum were affected less by stress than were the other inocula. Spore production was in general greater on sorghum than on maize, but the host effect varied amongst the inocula.     

Physiological description of multivariate interdependencies between process parameters,morphology and physiology during fed‐batch penicillin production

Optimization of productivity and economics of industrial bioprocesses requires characterization of interdependencies between process parameters and process performance. In the case of penicillin production, as in other processes, process performance is often closely interlinked with the physiology and morphology of the organism used for production. This study presents a systematic approach to efficiently characterize the physiological effects of multivariate interdependencies between bioprocess design parameters (spore inoculum concentration, pO₂ control level and substrate feed rate), morphology, and physiology. Method development and application was performed using the industrial model process of penicillin production. Applying traditional, statistical bioprocess analysis, multivariate correlations of raw bioprocess design parameters (high spore inoculum concentration, low pO₂ control as well as reduced glucose feeding) and pellet morphology were identified. A major drawback of raw design parameter correlation models; however, is the lack of transferability across different process scales and regimes. In this context, morphological and physiological bioprocess modeling based on scalable physiological parameters is introduced. In this study, raw parameter effects on pellet morphology were efficiently summarized by the physiological parameter of the biomass yield per substrate. Finally, for the first time to our knowledge, the specific growth rate per spore was described as time‐independent determinant for switching from pellet to disperse growth during penicillin production and thus introduced as a novel, scalable key process parameter for pellet morphology and process performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:689–699, 2014     

A Confocal microscopic study on colony morphology and sporulation of Bacillus sp.

Hexavalent chromium Cr(VI) is one of the dominant oxidation states of chromium that exist in the environment and is highly toxic to all forms of life. In the present study, we employ a confocal laser scanning microscope (CLSM) and investigate the effect of Cr(VI) on colony morphology of a Bacillus sp. isolated from soil exposed to tannery effluent. The colonies grown at chromium concentrations, control and 100 ppm are found to be opaque and beyond 200 ppm the colonies were translucent thus exhibiting phase variation. CLSM studies on colonies grown on control plates showed significant increase in height and in biovolume as a function of time whereas, the translucent colonies showed very little change in height and biovolume corresponding to the colony growth. Exopolymeric substance (EPS) content of translucent colonies was lesser than that of opaque colonies, indicating that EPS also plays a role in the observed phenomenon of phase variation. Studies on the effect of Cr(VI) on spore formation showed that Cr(VI) concentrations up to 100 ppm favored spore formation, while concentrations beyond 100 ppm showed a steady decline in spore formation.     

Effects of validamycin A on the morphology, growth and sporulation of Rhizoctonia cerealis, Fusarium culmorum and other fungi

All Basidiomycotina screened were sensitive to validamycin A, whereas most Ascomycotina and all Mucorales and Oomycetes were insensitive. Studies with Rhizoctonia cerealis and Fusarium culmorum showed that, in semi-solid culture, the antibiotic caused a decrease in colony radial growth rate and that this was associated with a decrease in mean hyphal extension rate and an increase in hyphal branching. However, the antibiotic did not alter the morphology of R. cerealis grown in liquid culture (shaken or stationary). Validamycin A caused a reduction in the number and viability of conidia produced by F. culmorum.     

Stochastic tunneling in the colonization of mutualistic organisms: Primary succession by mycorrhizal plants

In mutualism under spatial structure, asynchrony between the dispersions of the interacting species can be a key determinant of their dynamics. We focused on the plant-mycorrhizal fungi system to theoretically analyze the colonization process by calculating the probability of colony establishment under environmental fluctuation. This can be considered a joint process of two sub-processes before and after the association between the host plant and the mycorrhizal fungi in a novel habitat. When colony growth undergoes environmental fluctuation, the dynamics of colony size can be considered a combination of the two stochastic sub-processes that mediated the association event between the plant and the fungi. Therefore, properties of whole system are influenced by five parameters, means and variances of colony growth rates of two sub-systems, and a rate of association of plant and fungi. For the successful establishment of a colony, the second sub-process must start before the first sub-process finishes (i.e., extinction), which we refer to as “stochastic tunneling.” Our analysis of the establishment probability of a plant colony based on this concept revealed that (1) the mean colony growth rates of the host alone and the symbiotic association affect establishment probability in different ways, (2) the variance of colony growth rate of the symbiotic association reduces the establishment probability, although the variance of growth rate of the host alone facilitates the establishment probability when the mean growth rate of the host alone is negative, and (3) a trade-off between the mean colony growth rates of the host alone and the symbiotic association could result in the evolution of either a symbiotic or parasitic relationship, based on a host decision. The model we present is widely applicable to the colonization processes of both positive and negative species relationships, where the interacting species disperse independently.     

Phylogeny of certain biocontrol agents with special reference to nematophagous fungi based on RAPd

A number of phylogenetic studies have been carried out on biocontrol agents having similar biological control activity. However, no work has been carried out to determine the phylogenetic relationship amongst various groups of biological control agents with varied biocontrol properties. Our aim was to derive a phylogenetic relationship between diverse biocontrol agents belonging to the deuteromycetes and determine its correlation with their spore morphology and their biocontrol activity. RAPD was used to assess genomic variability in fungi used as biological control agents which included ten isolates of nematophagous fungi such as Arthrobotrys sp., Duddingtonia sp., Paecilomyces sp. and Verticillium sp., along with two isolates of fungal biocontrol agents such as Trichoderma sp. and two isolates of entomopathogenic fungi including Beauveria sp. A plant pathogenic fungus, Verticillium alboatrum was also included to increase the diversity of Deuteromycetes used. A similarity matrix was created using Jaccard's similarity coefficient & clustering was done using unweighted pair group arithmetic mean method (UPGMA). The final dendogram was created using a combination of two programs, Freetree and TreeExplorer. The phylogenetic tree constructed from the RAPD data showed marked genetic variability among different strains of the same species. The spore morphologies of all these fungi were also studied. The phylogenetic pattern could be correlated with the conidial and conidiophore morphology, a criterion commonly used for the classification of fungi in general and Deuteromycetes in particular. Interestingly, the inferred phylogeny showed no significant grouping based on either their biological control properties or the trapping structures amongst the nematophagous fungi as reported earlier by other workers. The phylogenetic pattern was also similar to the tree obtained by comparing the 18S rRNA sequences from the database. The result clearly indicates that the classical method of classification of these deuteromycete members on the basis of their spore morphology is reliable and could be used for identification of these fungi at species level. The PCR fragment pattern polymorphism exhibited by the various species of a genus and different strains of a species indicates that construction of probes from one or more of these fragments will prove to be useful as a rapid tool for identification of species and strains of nematophagous fungi in future.     

Screening fungi for synthesis of keratinolytic enzymes

Almost 300 common fungi were screened for synthesis of extracellular keratinases. About 54% of the fungi grew on agar plates with soluble keratin and excreted the enzymes. Some representatives of Fusarium , Acremonium and Geotrichum were the most active. However, when cultivating the promising strains in submerged conditions in a medium with porcine nail as the sole source of carbon and nitrogen, other fungi proved to be potent. Aspergillus flavus was the most powerful producer of extracellular keratinases, followed by Alternaria radicina , Trichurus spiralis and Stachybotrys atra.     

Production of phytotoxic spore germination liquids by Alternaria brassicae and A. brassicicola and their effect on species of the family Brassicaceae

Experiments assessed the susceptibility of Brassica spp. and non-Brassica spp. in the family Brassicaceae to infection by Alternaria brassicae and A. brassicicola, and determined the sensitivity of the host species to spore germination liquids (SGLs) produced by the pathogens on B. napus leaves. There was a wide range of sensitivity to the pathogens. Brassica spp. were generally more susceptible, and some non-Brassica spp. (Barbarea vulgaris and Capsella bursa-pastoris) were immune to A. brassicicola. Measurable damage was caused by SGLs but with significant variation between host species. Non-hosts and weak hosts also showed necrosis. It was concluded that, in the case of both pathogens, the toxic factors in these SGLs were host-selective. Selectivity in toxin production was also demonstrated in relation to the host surface or growing medium in which spores germinated. A substantial amount of toxin was produced on all Brassicaceae tested but not on unrelated species (Triticum aestivum, Pisum sativum and Lycopersicon esculentum). Neither pathogen produced measurable amounts of toxin when cultured in Czapek (Dox) broth.