Mohamed A. Moslem

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

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.

Colletotrichum species from Jasmine (Jasminum sambac)

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.

Fungi—an unusual source for cosmetics

Fruiting bodies of some wild and cultivatable mushrooms contain medicinal compounds which are being used in traditional medicines and cosmetics. There are numerous potential medicinal products from mushrooms that could be used in cosmeceuticals (products applied topically, such as creams, lotions, and ointments) or nutricosmetics (products that are ingested orally). This paper provides a review of the fungi presently used in cosmeceuticals and nutricosmetics with some examples of cosmetic types and products. Species presently used, or patented to be used, in cosmeceuticals and nutricosmetics include Agaricus subrufescens (= A. blazei, A. brasiliensis) Choiromyces maeandriformis Cordyceps sinensis, Ganoderma lucidum, Grifola frondosa, Hypsizygus ulmarium, Inonotus obliquus, Lentinula edodes, Polyporus spp., Trametes versicolor, Tremella fuciformis, Tuber spp., Schizophyllum commune and many other lesser used taxa. Cosmetics incorporating fungi include those for skin care such as anti-aging, anti-oxidants, skin revitalizing, skin whitening and hair products. The mushrooms presently used are traditionally known to produce medicinal compounds and thus were the first to be incorporated in cosmetic applications. There are, however, numerous other mushroom species that are untested, undescribed or not yet cultivatable and that have huge potential for use in the cosmetic industry. Some fungi are also used in biotransformation and the products such as lactic acid and ceramides could potentially be used in cosmetics.

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

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.

Biodegradation of diesel fuel hydrocarbons by mangrove fungi from Red Sea Coast of Saudi Arabia.

Mangrove sediments were collected from major mangrove stands on the Red Sea Coast of Saudi Arabia. Forty five isolates belonging to 12 genera were purified and five isolates as well as their consortium were found to be able to grow in association with petroleum oil as sole carbon source under in vitro conditions. The isolated strains were identified based on internal transcribed spacer (ITS) rDNA sequence analysis. The fungal strains with the greatest potentiality to degrade diesel oil, without developing antagonistic activity, were identified as Alternaria alternata, Aspergillus terreus, Cladosporium sphaerospermum, Eupenicillium hirayamae and Paecilomyces variotii. As compared to the controls, these fungi accumulated significantly higher biomass, produced extracellular enzymes and liberated larger volumes of CO2. These observations with GC–MS data confirm that these isolates displayed rapid diesel oil bioremoval and when used together as a consortium, there was no antagonistic activity.

An efficient method for DNA extraction from Cladosporioid fungi.

We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A(260)/A(280) ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyses.

Culture collections, the new herbaria for fungal pathogens

This paper discusses the importance of culture collections in plant pathology and reviews the methods currently available to store cultures. The preservation and maintenance of plant pathogenic fungi in a viable yet stable state for long periods has always been important, because isolates of these fungi can serve as standards for identification of quarantine taxa. Such isolates are also important for testing disease resistance and for plant breeding programs. The increasing use of molecular sequences analysis in the systematics of plant pathogenic fungi has meant that maintaining fungi in culture collections has become essential. Herein we discuss trends in the identification of plant and post-harvest pathogens, using Aspergillus, Colletotrichum, Phyllosticta and Mycosphaerella and its anamorphs as examples. Herbarium specimens, although still a requirement of the Botanical Code when describing new species are, perhaps, less important in providing useful information when defining a pathogenic species. Many pathogen groups consist of complexes of species and morphology alone can no longer distinguish among species. However, ex-type living cultures are essential for identification and future species comparisons that incorporate the use of molecular techniques. As such, ex-type cultures of any new species of pathogen, or when new diseases are reported or studies involving pathogenic strains are published, cultures of the taxa studied should be deposited in widely available culture collections, preferably in at least three members of World Federation for Culture Collections. Methods for the storage of fungal cultures such as water preservation, freezing, mineral oil overlay, freeze drying and lyophilization are reviewed in this paper. The main objective of culture preservation is to maintain the vigor and genetic characteristics of a pure culture. Therefore, safe long-term preservation methods are required to ensure fungal survival and retention of any valuable characteristics. To minimize the risk of any morphological, physiological, or genetic changes, several different preservation conditions should be used whenever possible. The present review also describes a complete preservation methodology that can be used for plant pathogenic fungi.

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

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 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 μL, as compared to 10 pg/25 μL 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.

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil.

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.

Molecular detection of ochratoxigenic Aspergillus species isolated from coffee beans in Saudi Arabia

Ten fungal isolates from coffee beans were morphologically identified as Aspergillus niger, A. ochraceus and A. carbonari-us (N = 5, 3, and 2, respectively). Only one isolate, morphologically identified as A. niger, was unable to produce ochratoxin A (OTA). This may be a new species in the Aspergillus section Nigri. OTA levels in all the other isolates were above the limit of detection (0.15 mg/kg). Based on microsatellite-primed PCR (MP-PCR) profiles, using three microsatellite primers, three main groups were obtained by UPGMA cluster analysis: A. niger, A. ochraceus and A. carbonarius. A clear-cut association was found between the MP-PCR genotype and the ability to produce OTA. Using the primer pairs OCRA1/OCRA2, a single fragment of about 400 bp was amplified only when genomic DNA from the A. ochraceus isolates was used.