Alternaria section Alternaria: Species, formae speciales or pathotypes?

The cosmopolitan fungal genus Alternaria consists of multiple saprophytic and pathogenic species. Based on phylogenetic and morphological studies, the genus is currently divided into 26 sections. Alternaria sect. Alternaria contains most of the small-spored Alternaria species with concatenated conidia, including important plant, human and postharvest pathogens. Species within sect. Alternaria have been mostly described based on morphology and / or host-specificity, yet molecular variation between them is minimal. To investigate whether the described morphospecies within sect. Alternaria are supported by molecular data, whole-genome sequencing of nine Alternaria morphospecies supplemented with transcriptome sequencing of 12 Alternaria morphospecies as well as multi-gene sequencing of 168 Alternaria isolates was performed. The assembled genomes ranged in size from 33.3-35.2 Mb within sect. Alternaria and from 32.0-39.1 Mb for all Alternaria genomes. The number of repetitive sequences differed significantly between the different Alternaria genomes; ranging from 1.4-16.5 %. The repeat content within sect. Alternaria was relatively low with only 1.4-2.7 % of repeats. Whole-genome alignments revealed 96.7-98.2 % genome identity between sect. Alternaria isolates, compared to 85.1-89.3 % genome identity for isolates from other sections to the A. alternata reference genome. Similarly, 1.4-2.8 % and 0.8-1.8 % single nucleotide polymorphisms (SNPs) were observed in genomic and transcriptomic sequences, respectively, between isolates from sect. Alternaria, while the percentage of SNPs found in isolates from different sections compared to the A. alternata reference genome was considerably higher; 8.0-10.3 % and 6.1-8.5 %. The topology of a phylogenetic tree based on the whole-genome and transcriptome reads was congruent with multi-gene phylogenies based on commonly used gene regions. Based on the genome and transcriptome data, a set of core proteins was extracted, and primers were designed on two gene regions with a relatively low degree of conservation within sect. Alternaria (96.8 and 97.3 % conservation). Their potential discriminatory power within sect. Alternaria was tested next to nine commonly used gene regions in sect. Alternaria, namely the SSU, LSU, ITS, gapdh, rpb2, tef1, Alt a 1, endoPG and OPA10-2 gene regions. The phylogenies from the two gene regions with a relatively low conservation, KOG1058 and KOG1077, could not distinguish the described morphospecies within sect. Alternaria more effectively than the phylogenies based on the commonly used gene regions for Alternaria. Based on genome and transcriptome comparisons and molecular phylogenies, Alternaria sect. Alternaria consists of only 11 phylogenetic species and one species complex. Thirty-five morphospecies, which cannot be distinguished based on the multi-gene phylogeny, are synonymised under A. alternata. By providing guidelines for the naming and identification of phylogenetic species in Alternaria sect. Alternaria, this manuscript provides a clear and stable species classification in this section.

Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes.

In the last two decades major changes have occurred in how microbial ecologists study microbial communities. Limitations associated with traditional culture-based methods have pushed for the development of culture-independent techniques, which are primarily based on the analysis of nucleic acids. These methods are now increasingly applied in food microbiology as well. This review presents an overview of current community profiling techniques with their (potential) applications in food and food-related ecosystems. We critically assessed both the power and limitations of these techniques and present recent advances in the field of food microbiology attained by their application. It is unlikely that a single approach will be universally applicable for analyzing microbial communities in unknown matrices. However, when screening samples for well-defined species or functions, techniques such as DNA arrays and real-time PCR have the potential to overtake current culture-based methods. Most importantly, molecular methods will allow us to surpass our current culturing limitations, thus revealing the extent and importance of the 'non-culturable' microbial flora that occurs in food matrices and production.

SodERF3, a novel sugarcane ethylene responsive factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants.

The molecular signals and pathways that govern biotic and abiotic stress responses in sugarcane are poorly understood. Here we describe SodERF3, a sugarcane (Saccharum officinarum L. cv Ja60-5) cDNA that encodes a 201-amino acid DNA-binding protein that acts as a transcriptional regulator of the ethylene responsive factor (ERF) superfamily. Like other ERF transcription factors, the SodERF3 protein binds to the GCC box, and its deduced amino acid sequence contains an N-terminal putative nuclear localization signal (NLS). In addition, a C-terminal short hydrophobic region that is highly homologous to an ERF-associated amphiphilic repression-like motif, typical for class II ERFs, was found. Northern and Western blot analysis showed that SodERF3 is induced by ethylene. In addition, SodERF3 is induced by ABA, salt stress and wounding. Greenhouse-grown transgenic tobacco plants (Nicotiana tabacum L. cv. SR1) expressing SodERF3 were found to display increased tolerance to drought and osmotic stress.

Root and Foot Rot on Tomato Caused by Phytophthora infestans Detected in Belgium.

In January 2003, a severe root and foot rot was observed on 2-month-old wilted tomato (Lycopersicon esculentum Mill.) plants in a large-scale (2.5 ha) commercial greenhouse setting in Belgium. Tomato plants (10%) produced from healthy nursery-grown seedlings and planted to new, clean rockwool and drip irrigation with UV-disinfected water developed symptoms. Symptom development was restricted to lower plant parts with severe rotting of the entire root system and dark lesions girdling the stem base. No symptoms of disease were observed on the foliage or upper stems. Cross sections of the stem base revealed brown discoloration of internal tissue, including the vascular tissue and pith. Dark brown lesions also occurred on the roots. Sections of the stem base, the upper roots (sampled near to the stem base), and the lower roots (sampled on roots deeper in the rockwool) were plated separately on corn meal agar. The oomycete pathogen Phytophthora infestans (Mont.) de Bary was identified in each sample on the basis of morphological characteristics observed directly with light microscopy. Branched sporangiophores with slight swellings and characteristic lemon-shaped sporangia (35 × 20 µm and ratio length/width of 1.75 µm) at their tips were obvious after incubation in darkness at 24°C. Oospores and chlamydospores were not observed. After multiple soil treatment with oomycete-specific fungicides, the plants recovered. Since the occurrence of P. infestans on roots is unusual, the identity of the pathogen on the diseased plant tissues was confirmed with three techniques, DNA array identification, internal transcribed spacer (ITS) sequencing, and a polymerase chain reaction (PCR) amplification using P. infestans-specific primers. DNA was directly processed from separate samples of upper and lower root and stem base tissue. The DNA array used was originally developed to detect and identify the key fungal pathogens of tomato (2). Among detector probes for other tomato pathogens, this array contains oligonucleotide detector probes for P. infestans (PIN1: 5'-GGT TGT GGA CGC TGC TAT T and PIN2: 5'-AAT GGA GAA ATG CTC GAT TC). These probes are based on ITS sequences (ITS I and ITS II). Using conserved ribosomal primers OOMUP18Sc (5'-TGC GGA AGG ATC ATT ACC ACA C) and ITS4, oomycete DNA was amplified by PCR and simultaneously labeled with alkaline-labile digoxigenin (2). All generated amplicons strongly hybridized to the oligonucleotide detector probes for P. infestans and not to any other pathogen-specific detector probe present on the array. The pathogen could not be detected in roots and stem bases of symptomless plants. In addition, the ITS-region was sequenced and showed 100% homology with multiple GenBank accessions of P. infestans sequences. As a third confirmatory test, a PCR was performed on DNA extracts from infected root and stem base tissues using a primer set specific to P. infestans (O8-3/O8-4 [1]). A band of the expected size was produced for the infected stem base and root samples. Until now, this pathogen was known worldwide to cause late blight on potatoes and tomatoes. To our knowledge, this is the first report of root and foot rot of tomato caused by P. infestans. References: (1) H. S. Judelson and P. W. Tooley. Phytopathology 90:1112, 2000. (2) B. Lievens et al. FEMS Microbiol. Lett. 223:113, 2003.

Rapid detection and identification of tomato vascular wilt pathogens using a DNA array.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or V. dahliae, are devastating diseases of tomato (Lycopersicon esculentum Mill.) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We demonstrate that by using this array these pathogens can be detected within 24 h from complex substrates like soil, plant material, and samples as they are collected by tomato growers in their greenhouses.