Showing papers by "Ali H. Bahkali published in 2011"

The genus Phomopsis: biology, applications, species concepts and names of common phytopathogens

Abstract: The genus Phomopsis (teleomorph Diaporthe) comprises phytopathologically important microfungi with diverse host associations and a worldwide distribution. Species concepts in Phomopsis have been based historically on morphology, cultural characteristics and host affiliation. This paper serves to provide an overview of the current status of the taxonomy in Phomopsis with special reference to biology, applications of various species, species concepts, future research perspectives and names of common pathogens, the latter being given taxonomic reappraisal. Accurate species identification is critical to understanding disease epidemiology and in developing effective control measures for plant diseases. Difficulties in accurate species identification using morphology have led to the application of alternative approaches to differentiate species, including virulence and pathogenicity, biochemistry, metabolites, physiology, antagonism, molecular phylogenetics and mating experiments. Redefinition of Phomopsis/Diaporthe species has been ongoing, and some species have been redefined based on a combination of molecular, morphological, cultural, phytopathological and mating type data. Rapid progress in molecular identification has in particular revolutionized taxonomic studies, providing persuasive genetic evidence to define the species boundaries. A backbone ITS based phylogenetic tree is here in generated using the sequences derived from 46 type, epitype cultures, and vouchers and is presented as a rough and quick identification guide for species of Phomopsis. The need for epitypification of taxonomic entities and the need to use multiple loci in phylogenies that better reflect species limits are suggested. The account of names of phytopathogens currently in use are listed alphabetically and annotated with a taxonomic entry, teleomorph, associated hosts and disease symptoms, including brief summaries of taxonomic and phylogenetic research. Available type culture information and details of gene sequences derived from type cultures are also summarized and tabulated.

Pestalotiopsis—morphology, phylogeny, biochemistry and diversity

Abstract: The genus Pestalotiopsis has received considerable attention in recent years, not only because of its role as a plant pathogen but also as a commonly isolated endophyte which has been shown to produce a wide range of chemically novel diverse metabolites. Classification in the genus has been previously based on morphology, with conidial characters being considered as important in distinguishing species and closely related genera. In this review, Pestalotia, Pestalotiopsis and some related genera are evaluated; it is concluded that the large number of described species has resulted from introductions based on host association. We suspect that many of these are probably not good biological species. Recent molecular data have shown that conidial characters can be used to distinguish taxa; however, host association and geographical location is less informative. The taxonomy of the genera complex remains confused. There are only a few type cultures and, therefore, it is impossible to use gene sequences in GenBank to clarify species names reliably. It has not even been established whether Pestalotia and Pestalotiopsis are distinct genera, as no isolates of the type species of Pestalotia have been sequenced, and they are morphologically somewhat similar. When selected GenBank ITS accessions of Pestalotiopsis clavispora, P. disseminata, P. microspora, P. neglecta, P. photiniae, P. theae, P. virgatula and P. vismiae are aligned, most species cluster throughout any phylogram generated. Since there appears to be no living type strain for any of these species, it is unwise to use GenBank sequences to represent any of these names. Type cultures and sequences are available for the recently described species P. hainanensis, P. jesteri, P. kunmingensis and P. pallidotheae. It is clear that the important species in Pestalotia and Pestalotiopsis need to be epitypified so that we can begin to understand the genus/genera. There are numerous reports in the literature that various species produce taxol, while others produce newly discovered compounds with medicinal potential and still others cause disease. The names assigned to these novel compound-producing taxa lack an accurate taxonomic basis, since the taxonomy of the genus is markedly confused. Until the important species have been epitypified with living strains that have been sequenced and deposited in public databases, researchers should refrain from providing the exact name of species.

Cochliobolus : an overview and current status of species

Abstract: The genus Cochliobolus (anamorphs Bipolaris, Curvularia) comprises many destructive plant pathogens that cause severe crop losses worldwide. The taxonomy of Cochliobolus is confused as frequent nomenclatural changes have occurred in the sexual and asexual states of species over the past 50 years. We provide an overview of these nomenclatural changes together with a morphological circumscription of the genus. Taxonomic notes and information about the life history of 55 species epithets of Cochliobolus listed in Index Fungorum are also given. Further information is given concerning the location of type cultures; availability of DNA sequence data derived from type cultures; mode of life; novel metabolite production; and use of Cochliobolus species in biocontrol. We provide a multilocus phylogenetic tree based on DNA sequence data derived from 25 ex-type and authentic cultures that shows the group as monophyletic. This paper represents the first comprehensive overview of Cochliobolus since 1987, including a summary of applications of species and molecular phylogenetic research. The 55 species of Cochliobolus are listed alphabetically, with synonyms, hosts and diseases, brief notes concerning taxonomic and phylogenetic research.

Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range.

Abstract: Plant pathogenic fungi adapt quickly to changing environments including overcoming plant disease resistance genes. This is usually achieved by mutations in single effector genes of the pathogens, enabling them to avoid recognition by the host plant. In addition, horizontal gene transfer (HGT) and horizontal chromosome transfer (HCT) provide a means for pathogens to broaden their host range. Recently, several reports have appeared in the literature on HGT, HCT and hybridization between plant pathogenic fungi that affect their host range, including species of Stagonospora/Pyrenophora, Fusarium and Alternaria. Evidence is given that HGT of the ToxA gene from Stagonospora nodorum to Pyrenophora tritici-repentis enabled the latter fungus to cause a serious disease in wheat. A nonpathogenic Fusarium species can become pathogenic on tomato by HCT of a pathogenicity chromosome from Fusarium oxysporum f.sp lycopersici, a well-known pathogen of tomato. Similarly, Alternaria species can broaden their host range by HCT of a single chromosome carrying a cluster of genes encoding host-specific toxins that enabled them to become pathogenic on new hosts such as apple, Japanese pear, strawberry and tomato, respectively. The mechanisms HGT and HCT and their impact on potential emergence of fungal plant pathogens adapted to new host plants will be discussed.

From morphology to molecular biology: can we use sequence data to identify fungal endophytes?

Abstract: Isolation followed by morphological identification was the traditional basis of all earlier endophyte studies. However, the use of molecular phylogenetics has become increasingly common in the identification of fungal endophytes, and during the period of 2007–2010 there were approximately 200 publications that reported data obtained using this approach. This new methodology involves using sequence data from isolates or whole DNA from plant substrates, which are amplified using fungus- specific primers. The data obtained are compared with sequences downloaded from public databases such as GenBank and then used to construct phylogenetic trees. The major problem with this approach is that much of the sequence data in these databases has been shown to be from isolates that were incorrectly named. In some species, as much as 86% of the sequences available are not from the organism whose name has been applied to the sequence in question. The use of these GenBank sequences to identify endophytic isolates by sequence similarity simply perpetuates the problem of wrong species identification, and any lists of endophytes established by such methods are likely to be highly erroneous. It is recommended that comparisons of sequence data be made using sequences from type species, and if such sequences are not available, then the data must be treated with caution.

Phyllosticta— an overview of current status of species recognition

Abstract: Phyllosticta is an important coelomycetous plant pathogenic genus known to cause leaf spots and various fruit diseases worldwide on a large range of hosts. Species recognition in Phyllosticta has historically been based on morphology, culture characters and host association. Although there have been several taxonomic revisions and enumerations of species, there is still considerable confusion when identifying taxa. Recent studies based on molecular data have resolved some cryptic species and some novel taxa have been discovered. However, compared to the wide species diversity and taxonomic records, there is a lack of molecular studies to resolve current names in the genus. A phylogenetic tree is here generated by combined gene analysis (ITS, partial actin and partial elongation factor 1α) using a selected set of taxa including type-derived sequences available in GenBank. Life modes, modal lifecycle and applications of the genus in biocontrol and metabolite production are also discussed. We present a selected set of taxa as an example of resolved and newly described species in the genus and these are annotated with host range, distribution, disease symptoms and notes of additional information with comments where future work is needed.

A reappraisal of Microthyriaceae

Abstract: The family Microthyriaceae sensu Lumbsch and Huhndorf 2010 is a poorly known but interesting family comprising 50 genera consisting of foliar epiphytes or saprobes on dead leaves and stems. We re-visited the family based on examinations of generic types where possible. Members are distributed in Aulographaceae, Asterinaceae, Microthyriaceae, Micropeltidaceae and Palmulariaceae and notes are provided on each of these families. Nine genera are transferred from Microthyriaceae to Asterinaceae, and two to Aulographaceae based on the splitting or dissolving nature of the thyriothecia to release ascospores. New sequence data for a number of species and genera are provided. Microthyriaceous members growing on other fungi and lichens differ from Microthyriaceae sensu stricto and the family Trichothyriaceae is reinstated to accommodate these taxa. Other genera of Microthyriaceae belong in Rhytismataceae, Stictidaceae, Venturiales incertae cedis, Dothideomyetes genera incertae cedis,Hypocreales incertae cedis and Ascomycota genera incertae cedis. The family Microthyriaceae is reduced to seven genera characterised by superficial, flattened thyriothecia, with the cells of the upper wall radiating in parallel arrangement from the distinct central ostiolar opening, while the lower peridium is generally poorly developed. Sequence data is provided for five species with thyriothecia and Paramicrothyrium and Neomicrothyrium are described as new genera and Micropeltis zingiberacicola is introduced as a new species. Our phylogenetic analysis underscores the high genetic diversity for thyriotheciate species and there is no clear clade that can be well defined as Microthyriales. Nuclear ribosomal data support multiple polyphyletic lineages within Microthyriaceae and Micropeltidaceae. Some unexpected DNA based phylogenetic relationships such as those between Muyocopron and Saccardoella will require corroboration with more complete taxon sampling as well as additional non ribosomal markers. There are few differences between Aulographaceae, Asterinaceae and Palmulariaceae and these families may need synonymising.

A molecular, morphological and ecological re-appraisal of Venturiales―a new order of Dothideomycetes

Abstract: The Venturiaceae was traditionally assigned to Pleosporales although its diagnostic characters readily distinguish it from other pleosporalean families. These include a parasitic or saprobic lifestyle, occurring on leaves or stems of dicotyledons; small to medium-sized ascomata, often with setae; deliquescing pseudoparaphyses; 8-spored, broadly cylindrical to obclavate asci; 1-septate, yellowish, greenish or pale brown to brown ascospores; and hyphomycetous anamorphs. Phylogenetically, core genera of Venturiaceae form a monophyletic clade within Dothideomycetes, and represent a separate sister lineage from current orders, thus a new order—Venturiales is introduced. A new family, Sympoventuriaceae, is introduced to accommodate taxa of a well-supported subclade within Venturiales, which contains Sympoventuria, Veronaeopsis simplex and Fusicladium-like species. Based on morphology and DNA sequence analysis, eight genera are included in Venturiaceae, viz. Acantharia, Apiosporina (including Dibotryon), Caproventuria, Coleroa, Pseudoparodiella, Metacoleroa, Tyrannosorus and Venturia. Molecular phylogenetic information is lacking for seven genera previously included in Venturiales, namely Arkoola, Atopospora, Botryostroma, Lasiobotrys, Trichodothella, Trichodothis and Rhizogenee and these are discussed, but their inclusion in Venturiaceae is doubtful. Crotone, Gibbera, Lineostroma, Phaeocryptopus, Phragmogibbera, Platychora, Polyrhizon, Rosenscheldiella, Uleodothis and Xenomeris are excluded from Venturiales, and their ordinal placement needs further investigation. Zeuctomorpha is treated as a synonym of Acantharia.

Revision of lignicolous Tubeufiaceae based on morphological reexamination and phylogenetic analysis

Abstract: In this paper we revisit the family Tubeufiaceae with notes on genera that we have re-examined where possible. Generic type specimens of Acanthophiobolus, Kamalomyces, Podonectria, Thaxteriella and Thaxteriellopsis were re-examined, described and illustrated and shown to belong to Tubeufiaceae. Notes are provided on Acanthostigma, Chaetosphaerulina, Thaxterina and Tubeufia, which are retained in Tubeufiaceae; however, we were unable to locate the types of these genera during the time frame of this study. Allonecte is excluded from the Tubeufiaceae, as the ascospores are fusiform-ellipsoidal, grey-brown and 1-septate and the asci are cylindrical, all of which are features more typical of Pleosporaceae, where it is transferred. Byssocallis has yellow to orange ascomata and clavate ascospores which is atypical of Tubeufiaceae. Thus its taxonomic status needs to be reevaluated. Lentendraeopsis has an endophytic habit, cylindro-clavate asci and two-celled ascospores more typical of Pleosporales, where it is transferred. Taphrophila has small ascomata, a thin peridium, branching setae around the apex of the ascomata, clavate to saccate asci and lacks pseudoparaphyses. These are features atypical of the Tubeufiaceae, and Taphrophila should be placed in the Dothideomycetes incertae cedis. Twelve new collections of Tubeufiaceae from Thailand were isolated, and their DNA was extracted. The sequence data of LSU, SSU and ITS rDNA were amplified and analyzed using parsimony and likelihood methods. The results of phylogenetic analysis was used to establish the inter-generic relationships in Tubeufiaceae. Thaxteriellopsis lignicola, epitypified in this investigation, is a sister taxon in the family Tubeufiaceae based on phylogenetic analysis of rRNA sequence data. Chlamydotubeufia is introduced as a new genus based on the production of dictyochlamydosporous anamorphs, including two new species. Three new species, one each in Acanthostigma, Tubeufia and Thaxteriella are also described and illustrated. The phylogenetic placement of these genera is also discussed.

Capnodiaceae

Abstract: In this paper we revisit the Capnodiaceae with notes on selected genera. Type specimens of the ascomycetous genera Aithaloderma, Anopeltis, Callebaea, Capnodaria, Echinothecium, Phragmocapnias and Scorias were re-examined, described and illustrated. Leptoxyphium is anamorphic Capnodiaceae and Polychaeton is a legitimate and earlier name for Capnodium, but in order to maintain nomenclatural stability we propose that the teleomorphic name should be considered for the approved lists of names currently in preparation for fungi. Notes are provided on the ascomycetous genus Scoriadopsis. However, we were unable to locate the type of this genus during the time frame of this study. The ascomycetous genera Aithaloderma, Ceramoclasteropsis, Hyaloscolecostroma and Trichomerium are excluded from Capnodiaceae on the basis of having ascostromata and trans-septate hyaline ascospores and should be accommodated in Chaetothyriaceae. Callebaea is excluded as the ascomata are thyriothecia and the genus is placed in Micropeltidaceae. Echinothecium is excluded as synonym of Sphaerellothecium and is transferred to Mycosphaerellaceae. The type specimen of Capnophaeum is lost and this should be considered as a doubtful genus. The coelomycetous Microxiphium is polyphyletic, while the status of Fumiglobus, Polychaetella and Tripospermum is unclear. Fourteen new collections of sooty moulds made in Thailand were isolated and sequenced. The nuclear large and small rDNA was partially sequenced and compared in a phylogeny used to build a more complete understanding of the relationships of genera in Capnodiaceae. Four new species are described and illustrated, while Phragmocapnias and Scorias are epitypified with fresh collections.

Astrosphaeriella is polyphyletic, with species in Fissuroma gen. nov., and Neoastrosphaeriella gen. nov.

Abstract: Collections of fungi from bamboo and palm plants in Thailand resulted in the identification of several species of Astrosphaeriella, including the type species A. fusispora, which is a synonym of A. stellata. Species of Astrosphaeriella have been previously circumscribed on the basis of morphology and, to a lesser extent, on host affiliation. In order to obtain a phylogenetic understanding of the genus, eleven strains of Astrosphaeriella sensu lato were sequenced in this study. Molecular analyses based on a combined dataset of 18S and 28S nrDNA sequences were carried out to infer the phylogenetic placement of these strains in the Pleosporales. The phylogenetic analyses showed that Astrosphaeriella is polyphyletic, with Astrosphaeriella species clustering in four clades, two clades, including species with slit-like ostioles, clustered in Aigialaceae; the clade that includes the generic type clustered together with Delitschia; and A. Africana, which has striate ascospores, deviated from these three clades and had a basal position in the Pleosporales. A new combination in Fissuroma gen. nov. and new genus Neoastrosphaeriella are introduced in Aigialaceae to include the species with slit-like ascomata.

Colletotrichum species from Jasmine (Jasminum sambac)

Abstract: Colletotrichum species associated with leaf and flower anthracnose of jasmine (Jasminum sambac) in the Ho Chi Minh region of Vietnam are reported. The disease of jasmine plantations was considered serious as it likely reduced flower yield. Leaves were colonized by Colletotrichum species which formed chlorotic regions with light brown necrotic centres, which eventually covered the whole leaf and subsequently caused defoliation and dieback and whole flowers were blighted. Nine strains of Colletotrichum species were isolated from diseased leaves and flowers and partial ITS rDNA sequences were analysed and morphologies compared across similar species. Based on ITS sequence analysis and morphological characters, three strains were identified as C. truncatum, while one strain was identified as C. siamense. The remaining five strains did not cluster with any known species for which type sequences are available and therefore partial actin (ACT), β-tubulin (TUB2), calmodulin (CAL), glutamine synthetase (GS), glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes of the isolates were sequenced. Based on the reconstructed multiloci molecular phylogeny, two taxa are formally introduced as new species. Another strain was not well resolved in the phylogenetic tree and herein described as Colletotrichum sp. Further studies are needed to prove its distinctiveness. The morphology and growth rate of all taxa are described and compared with similar species.

An Optimized Protocol for DNA Extraction from Wheat Seeds and Loop-Mediated Isothermal Amplification (LAMP) to Detect Fusarium graminearum Contamination of Wheat Grain

Abstract: A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In combination with the LAMP assay it is a fast and cost-effective alternative to traditional diagnostic methods for the early detection of toxigenic fusaria in cereals.

The need to carry out re-inventory of plant pathogenic fungi

Abstract: Plant pathogenic fungi have long been documented through concerted efforts of mycologists and plant pathologists; these records have served as the basis for regional and countrywide checklists which have since been put into databases listing hosts and associated fungi. They are used by governments and scientists to formulate trade quarantine policies and determine research funding, such as in plant breeding programs and disease control. With the ability to use molecular characters to study the systematics of fungi it is clear that morphologically defined species are often large complexes comprised of genetically and biologically distinct species. Use of molecular techniques to examine species complexes has revealed cryptic species in many important plant pathogenic genera, e.g. Botryosphaeria, Colletotrichum, Fusarium, and Mycosphaerella. It has occurred to such an extent that existing checklists and databases need updating. It is important that the data from these studies, including changes in taxonomy and nomenclature, be incorporated into the databases of plant pathogenic fungi to support accurate plant quarantine decisions. In addition, epitypifying fungi by re-collecting material from type habitats and isolating the organism into pure culture will provide essential materials for systematics studies to further clarify the taxonomy and phylogeny of plant pathogenic fungi. Overall, we conclude that disease lists are likely to be highly outdated and advocate the need for countrywide re-inventory of plant pathogens. As a result of these studies, tools can be developed that use morphological or molecular characters, or both, to promote accurate identification of plant pathogenic fungi.

Propyl Gallate Synthesis Using Acidophilic Tannase and Simultaneous Production of Tannase and Gallic Acid by Marine Aspergillus awamori BTMFW032

Abstract: Marine Aspergillus awamori BTMFW032, recently reported by us, produce acidophilic tannase as extracellular enzyme. Here, we report the application of this enzyme for synthesis of propyl gallate by direct transesterification of tannic acid and in tea cream solubilisation besides the simultaneous production of gallic acid along with tannase under submerged fermentation by this fungus. This acidophilic tannase enabled synthesis of propyl gallate by direct transesterification of tannic acid using propanol as organic reaction media under low water conditions. The identity of the product was confirmed with thin layer chromatography and Fourier transform infrared spectroscopy. It was noted that 699 U/ml of enzyme could give 60% solubilisation of tea cream within 1 h. Enzyme production medium was optimized adopting Box–Behnken design for simultaneous synthesis of tannase and gallic acid. Process variables including tannic acid, sodium chloride, ferrous sulphate, dipotassium hydrogen phosphate, incubation period and agitation were recognized as the critical factors that influenced tannase and gallic acid production. The model obtained predicted 4,824.61 U/ml of tannase and 136.206 μg/ml gallic acid after 48 h of incubation, whereas optimized medium supported 5,085 U/ml tannase and 372.6 μg/ml of gallic acid production after 36 and 84 h of incubation, respectively, with a 15-fold increase in both enzyme and gallic acid production. Results indicated scope for utilization of this acidophilic tannase for transesterification of tannic acid into propyl gallate, tea cream solubilisation and simultaneous production of gallic acid along with tannase.

Three new species of Lentinus from northern Thailand

Abstract: There have been few studies on the taxonomy and biodiversity of the genus Lentinus in Thailand, which is a genus of edible mushrooms. Recently, collections from 17 sites in northern Thailand yielded 47 specimens of Lentinus sensu lato. Three were shown to be new species of Lentinus sensu stricto and Lentinus roseus, L. concentricus and L. megacystidiatus are introduced in this paper. The new species are described and illustrated with line drawings and are justified and compared with similar taxa. Furthermore, ITS sequence data do not match closely with any species presently lodged in GenBank.

Stem cells: Biology and clinical potential

Abstract: Stem cell technology has developed rapidly in recent years to the point that we can now envisage its future use in a variety of therapeutic areas. This review seeks to summarize the types and sources of stem cells that may be utilized in this way, their pattern of development, their plasticity in terms of differentiation and transdifferentiation, their ability to self-renew, the privileged microenvironment in which they are housed, their cell surface markers used to track them, issues relating to their transfection, and their fate. Particular reference is made, as prime examples, to how both the function of mesenchymal and neural stem cells are being studied experimentally, and currently used clinically in certain circumstances, towards the ultimate aim of their mainstream therapeutic use. Key words: Stem cells, apoptosis, differentiation, mesenchymal and neural stem cells, therapy.

Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer

Abstract: Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola; no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 degrees C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 muL, as compared to 10 pg/25 muL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, specific, cost-effective and reliable for the identification and quantification of M. graminicola in wheat.

Freeze- and Thaw-Based Procedures for Extracting DNA from Activated Sludge

Abstract: Activated sludge treatment is one of the most popular biological processes for wastewater treatment. Detection of activated sludge structures from complex substrates such as soil and water is not easy. A rapid and effective method for extracting high molecular weight amplifiable DNA from activated sludge was modified. Five nanogram quantities of DNA per milligram activated sludge were recovered with SDS-based and freeze-and-thaw procedures. The ratio of absorbance at 260 nm to absorbance at 280 nm varies between 1.6 and 2, which is an indicator of the purity of DNA. Gel electrophoresis of the isolated DNA further showed intact genomic DNA bands of high molecular weight (greater than 12,000 base pairs) with no RNA contamination. The quality of obtained DNA was verified by PCR amplification reactions and restriction enzyme digestion. A comparison of the optimized protocol with commercial dBioZol DNA isolation kit suggested that the method described in this report would be more efficient in removing PCR inhibitors from activated sludge samples. The random amplified polymorphic DNA (RAPD) patterns demonstrated the genetic diversity of activated sludge samples. A 542-bp fragment of the 16S rRNA gene of the bacterial isolates was amplified. PCR using primers targeting 16S rDNA shows promise in the enumeration of gram-positive bacteria in activated sludge samples. The current protocol can be used to efficiently monitor the presence of microorganisms in sludge from effluent treatment plants.

Bio-fungicidal activity of aloe vera sap against mycotoxigenic seed-borne fungi

Abstract: Mycotoxigenic fungi isolated from corn, sorghum grains and peanut kernels were identified. Nine fungal species belonging to three genera were recovered from the tested samples. With the exception of the sorghum isolate, all tested Aspergillus flavus isolates were capable of pro- ducing variable amounts of G1 and B1 aflatoxin ranging from 1-6 parts per billion (ppb). The highest amount of aflatoxin B1 (8ppb) was produced by A. flavus isolated from corn grains. The sorghum isolate of Penicillium oxalicum was the highest producer of citreoviridin (37ppb). Fusa- rium subglutinans isolate recovered from popcorn was capable of producing fumonisin B1, zearalenone and vomitoxin (DON). Corn isolate of Fusarium proliferatum however, failed to produce fumonisin B1 and sorghum isolate of Fusarium verticillioides produced only fu- monisin B1. The antifungal activity of yellow liquid frac- tion of Aloe vera against toxigenic fungi was tested. All tested concentrations were effective in inhibiting fungal growth.

Development of di‐ and tetranucleotide repeat primer for discrimination of fusarium species

Abstract: The genetic diversity profiles of 19 isolates of the fungal pathogen Fusarium species, 14 from Egypt and 5 from Germany, were analyzed based on morphological characteristics and microsatellite markers. Five microsatellites were selected and primers were designed. Microsatellite-primed polymerase chain reaction using the dinucleotide and tetranucleotide primers showed clear polymorphisms among the different Fusarium spp. isolates. Both primers gave similar results in phenetic analysis of genetic similarity between populations. Between Fusarium spp. isolates, similarities ranged from 38 to 62 for interspecific and 62 to 94 for intraspecific comparisons. Two major groups were observed in the dendrogram, which was divided into three subgroups. One of them consisted the five F. oxysporum f. sp. vasinfectum isolates at the genetic similarity of 92. Isolates Fov1, Fov3 and Fov5 showed high genetic relatedness (100). With the second main cluster, at 62 similarity, one subcluster could be discerned; the subcluster contained F. oxysporum, F. solani, F. sambucinum, F. poae and Fusarium spp. isolates. These experiments reveal that microsatellite primers are reliable, sensitive and technically simple tools for assaying genetic variability in Fusaria.