First Report of Apple Bitter Rot Caused by Colletotrichum godetiae in the United Kingdom.

Apple is an important crop in United Kingdom, with a total production of 233,750 tonnes in 2011. Symptoms of apple bitter rot were observed on apple fruits (Malus domestica L.) in the Newcastle area, United Kingdom, in October 2008. Lesions were round, 1 to 5 cm in diameter, brown and dry, with acervuli producing yellowish spore masses in concentric bands. Infected material was sent to the W-HRI (University of Warwick) for identification of the causal agent. Fungal isolates with morphological characteristics similar to those of Colletotrichum acutatum sensu lato were isolated from diseased fruits. Monoconidial isolates were grown on PDA at 25°C with a 12-h light period. The cultures were light gray, with cottony aerial mycelium getting darker with age and with color ranging from whitish to dark gray on the reverse side of the colony. The cultures have yellowish spores masses and dark melanized structures similar to acervuli. Colletotrichum spp. are difficult to identify solely on morphology; therefore, representative isolates were used for multi-locus gene sequencing and characterization (1). Genomic DNA was extracted using a modified Chelex100 protocol. Three loci were amplified and sequenced: the ITS region was amplified and sequenced using the universal primers ITS4 and ITS5. Primers TB5 and TB6 were used for the amplification and sequencing of the variable region of the TUB gene. Primers GDF1 and GDR1 were used to amplify a 200-bp intron region of the GAPDH gene. No differences were found among the strains at any of the loci. One sequence for each locus has been deposited in GenBank under accessions KF834206 (ITS), KF834207 (TUB), and KF834208 (GAPDH). In GenBank, ITS sequences matched with 100% identity to C. higginsianum (EU400147) and to C. gloeosporioides (AJ301931 to 972); and with identity between 99.6 and 99.8% with sequences belonging to C. godetiae (part of C. acutatum species complex). The TUB sequences match with 100% identity to more than 25 sequences belonging to C. godetiae. The GAPDH sequences match with 100% identity to JQ948739 and 35 belonging to C. godetiae strains IMI 381927 and CBS 131331. A multilocus phylogenetic tree (ITS, TUB, and GAPDH) was reconstructed using sequences of reference strains belonging to C. higginsianum, C. gloeosporioides, C. godetiae, and related species. The phylogenetic tree confirmed the identity of the strains isolated from apple as C. godetiae. Koch's postulates were tested with representative isolate by artificial inoculation of 12 healthy fruits of the cv. Golden Delicious. Fruit surfaces were sterilized with 70% ethanol, wounded with a sterile needle, and then inoculated with a plug of actively growing mycelium prepared from a 10-day-old culture grown on PDA. Inoculated fruits were incubated in sterile conditions at 25°C with a 12-h photoperiod. In 83% of fruits, symptoms appeared between 7 and 15 days later. The rot begins as light brown, circular lesion getting darker with orange spore masses. Fungal colonies isolated from the lesions and cultured on PDA have identical morphological characteristics of the isolate used for the pathogenicity assay. To the best of our knowledge, this is the first report of apple bitter rot caused by C. godetiae in the United Kingdom. Apple bitter rot is spread worldwide and in moist, temperate regions it is considered one of the most important diseases causing considerable crop losses. Since the losses are more severe under prolonged warm and wet weather conditions, bitter rot caused by C. acutatum species may become an emerging problem in the United Kingdom in the near future, and may require investigation of management practices to control this new disease. References: (1) R. Baroncelli. Colletotrichum acutatum sensu lato: From diversity study to genome analysis. Coventry, United Kingdom, PhD thesis, 2012. (2) U. Damm et al. Stud. Mycol. 73:37, 2012.

Environmental regulation of reproductive phase change in Agaricus bisporus by 1-octen-3-ol, temperature and CO2.

Reproductive phase change from vegetative mycelium to the initiation of fruiting in Agaricus bisporus is regulated in large part by the sensing of environmental conditions. A model is proposed in which three separate environmental factors exert control at different stages of the reproductive developmental process change. The eight carbon volatile 1-octen-3-ol controls the early differentiation from vegetative hyphae to multicellular knots; temperature reduction is essential for the later differentiation of primodia; and carbon dioxide level exerts quantitative control on the number of fruiting bodies developed. Analysis of transcriptomic changes during the reproductive phase change was carried out with initiation-specific microarrays, and the newly published A. bisporus genome was used to analyse the promoter regions of differentially regulated genes. Our studies have shown there to be both early and late initiation responses relating to sensing of eight carbon volatiles and temperature respectively. A subset of 45 genes was transcriptionally regulated during the reproductive phase change which exhibited a range of functions including cell structure, nitrogen and carbon metabolism, and sensing and signalling. Three gene clusters linking increased transcription with developmental stage were identified. Analysis of promoter regions revealed cluster-specific conserved motifs indicative of co-ordinated regulation of transcription.

Magnaporthe oryzae populations adapted to finger millet and rice exhibit distinctive patterns of genetic diversity, sexuality and host interaction.

In this study, host-specific forms of the blast pathogen Magnaporthe oryzae in sub-Saharan Africa (SSA) were characterised from distinct cropping locations using a combination of molecular and biological assays. Finger millet blast populations in East Africa revealed a continuous genetic variation pattern and lack of clonal lineages, with a wide range of haplotypes. M. oryzae populations lacked the grasshopper (grh) element (96%) and appeared distinct to those in Asia. An overall near equal distribution (47-53%) of the mating types MAT1-1 and MAT1-2, high fertility status (84-89%) and the dominance of hermaphrodites (64%) suggest a strong sexual reproductive potential. Differences in pathogen aggressiveness and lack of cultivar incompatibility suggest the importance of quantitative resistance. Rice blast populations in West Africa showed a typical lineage-based structure. Among the nine lineages identified, three comprised ~90% of the isolates. Skewed distribution of the mating types MAT1-1 (29%) and MAT1-2 (71%) was accompanied by low fertility. Clear differences in cultivar compatibility within and between lineages suggest R gene-mediated interactions. Distinctive patterns of genetic diversity, sexual reproductive potential and pathogenicity suggest adaptive divergence of host-specific forms of M. oryzae populations linked to crop domestication and agricultural intensification.

Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles.

Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 10(4) conidia, with >70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles.

Differential expression of a putative riboflavin-aldehyde-forming enzyme (raf) gene during development and post-harvest storage and in different tissue of the sporophore in Agaricus bisporus.

Cloning and characterisation of a putative riboflavin-aldehyde-forming enzyme gene (raf) from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis are described. Three cDNA clones were isolated following differential screening of cDNA libraries from rapidly expanding sporophores and post-harvest stored sporophores. The cDNA sequence and predicted translation analysis revealed an open reading frame (ORF) of 348 nucleotides encoding a polypeptide of 115 amino acids, with three introns (56-66 bases) interrupting the genomic ORF. Blast X searches of the databases with the gene sequence showed homology (40% identity and 56% similarity) to the riboflavin-aldehyde-forming enzyme gene from Schizophyllum commune. In A.bisporus, the raf gene sequence upstream of the ORF contained a large CT-rich putative regulatory element (-64 to -24 bases) found in highly expressed genes in various mushrooms, and a 6-base motif present in the 3' end of the genomic sequence, but not in the corresponding 3' non-coding part of the cDNA, was identified. The raf gene transcripts increased abundantly in rapidly developing sporophores as well in post-harvest stored sporophores. Differential expression of the raf gene transcripts in different tissues of the sporophore was also observed, with higher levels in the stipe compared with the cap and gills. The temporal and spatial expression patterns observed suggest transcriptional regulation of the raf gene during A. bisporus morphogenesis.

Molecular and phenotypic analyses reveal association of diverse Colletotrichum acutatum groups and a low level of C. gloeosporioides with olive anthracnose.

Anthracnose (Colletotrichum spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of Colletotrichum spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128 Colletotrichum spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and beta-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed Colletotrichum acutatum to be dominant (>97%) with limited occurrence of Colletotrichum gloeosporioides (<3%). Among C. acutatum populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other C. acutatum groups and C. gloeosporioides were sporadic in their occurrence, mostly related to marginal areas of olive cultivation. C. gloeosporioides, isolated from olive fruits with symptoms indistinguishable from those of C. acutatum, showed same virulence rating as the most virulent C. acutatum isolate from group A2. C. acutatum and C. gloeosporioides isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from beta-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.

Genotypic and phenotypic diversity in Colletotrichum acutatum, a cosmopolitan pathogen causing anthracnose on a wide range of hosts.

SUMMARY Colletotrichum acutatum causes anthracnose on a wide range of hosts including woody and herbaceous crops, ornamentals, fruits and conifers. Almond, citrus, lupin, olive and strawberry are some of the crops in which C. acutatum diseases are economically important. With the application of molecular markers and diagnostic PCR over the last 10-15 years, C. acutatum was identified as a major pathogen on a number of hosts instead of or along with C. gloeosporioides. C. acutatum displays high levels of genotypic and phenotypic diversity. The global populations of this cosmopolitan pathogen fit into at least eight distinct molecular groups, A1-A8, which show some degree of correlation with the morphological characteristics and varying patterns of host association and geographical distribution. The pathogen has complex epidemiology, exhibiting pathogenic and non-pathogenic lifestyles on target hosts, non-target crops and weeds. C. acutatum populations also show pathogenic variability and cross-infection potential in relation to a number of hosts. Molecular genetic tools are being developed to investigate the pathogenicity mechanisms of this key pathogen. This article mainly focuses on the global population diversity in C. acutatum, pathogen epidemiology and diagnosis, host colonization processes, and the development of tools for the identification and analysis of genes associated with pathogenicity. Background information on the pathogen origin, host range, disease symptoms and disease management strategies is also provided.

Genetic and Morphological Characterization of Colletotrichum acutatum Causing Anthracnose of Lupins.

ABSTRACT Anthracnose, caused by Colletotrichum sp., is a serious problem of lupins (Lupinus spp.) worldwide. Morphological characters and molecular markers were used to characterize 43 Colletotrichum isolates from lupins, 8 isolates from other hosts, and 18 reference isolates representing related Colletotrichum spp., to assess the pathogen diversity and resolve its taxonomy. All lupin Colletotrichum isolates tested positive with C. acutatum-specific polymerase chain reaction (PCR) and did not test positive with C. gloeosporioides-specific PCR. Spore shape and colony diameter as well as insensitivity to benomyl grouped the lupin anthracnose isolates closer to C. acutatum than to C. gloeosporioides. Analysis of internal transcribed spacer (ITS) sequences of 57 Colletotrichum isolates grouped all lupin isolates with C. acutatum and distinct from C. gloeosporioides. Further, tub2 and his4 sequences revealed groups concordant with ITS, reducing the excessive dependence on the latter. Arbitrarily primed-PCR and amplified fragment length polymorphism analyses revealed intraspecific subgroups, but neither was useful to decipher species level relationships. ITS, tub2, and his4 results strongly support designating lupin anthracnose pathogen as C. acutatum or its subspecies. Most Colletotrichum isolates from lupins from worldwide locations are genetically homogeneous and form a distinct subgroup within C. acutatum. Present results also underline the potential of the C. acutatum-specific PCR for routine pathogen diagnosis.

Genetic diversity and interrelationships among common European Suillus species based on ribosomal DNA sequences.

The nucleotide sequence of the internal transcribed spacer (ITS) 1 and 2 regions was determined for 22 Suillus isolates representing seven species. Analysis of the sequence data along with 12 related sequences from databases divided the 34 isolates into seven groups, and six of these were supported by 100% bootstrap values. Isolates belonging to Suillus granulatus (0-11 divergence) were divided into three groups (SG1, SG2 and SG3); SG1 and SG2 had 100% bootstrap values. SG2 was clustered with two Suillus collinitus isolates and a Suillus variegatus isolate; SG3 was linked to a previously characterised Suillus subluteus isolate. These results clearly indicate heterogeneity among the isolates described under these species. Intra- (0.4-11) and inter- (2.4-13.6) specific divergence varied considerably. Suillus laricinus showed a high level (0.5-4.4) of intra-specific divergence. Suillus bovinus showed maximum divergence (11.4-13.3) to all other species examined. The potential of the ITS region for understanding the intra- and inter-specific relationships among Suillus species and refining their taxonomy is discussed.

DNA extraction method for PCR in mycorrhizal fungi.

AIMS: To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. METHODS AND RESULTS: The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. CONCLUSION: DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi.

Cloning and characterisation of a chitin synthase gene cDNA from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis.

Full-length cDNA of a chitin synthase gene (chs1) was cloned from Agaricus bisporus by screening a cDNA library with a PCR amplified fragment of the chitin synthase gene. The chs1 contains an open reading frame of 2727 bp encoding a polypeptide of 909 amino acids and deduced molecular mass 102.3 kDa and pI 8.23. The central region of chs1 showed strong homology to other fungal chitin synthase genes with seven conserved domains. It belongs to the chitin synthase class III, analogous to chsB from Aspergillus nidulans, chsC and chsG from A. fumigatus and chs-1 from Neurospora crassa. It appears to be fruit body induced as the transcripts were higher in the developing mushroom compared to any mycelial stage.

Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences.

The potential use of the ribosomal DNA internal transcribed spacer (ITS) sequences in understanding the phylogeny and systematics of Colletotrichum species has been evaluated. Sequence data from a limited number of isolates revealed that in Colletotrichum species the ITS 1 region (50.3% variable sites) shows a greater degree of intra- and inter-specific divergence than ITS 2 (12.4% variable sites). Nucleotide sequences of the ITS 1 region from 93 isolates representing 18 Colletotrichum species were determined. Data for 71 of these isolates where molecular and morphological identities concurred were used for phylogenetic analysis. The size of the ITS 1 region varied from 159 to 185 base pairs. Maximum intraspecific divergence was recorded with C. acutatum (5.8%), and C. capsici showed the greatest level of interspecific divergence (8.9-23.3%). Parsimony and distance analyses gave similar tree topologies. The bootstrapped consensus parsimony tree divided the 18 Colletotrichum species into six phylogenetic groups, designated 1-6. These groups, however, are not congruent with species clusterings based on spore shape. For example, the straight cylindrical spored species were represented both in groups 1 and 6; group 6 also included the falcate fusiform spored species C. capsici. The molecular evidence suggests refinement of the species concepts of some of the taxa examined. In group 6, divergence between C. gloeosporioides and C. fuscum (0.6-3.0%) or C. kahawae (0.6-3.0%) or C. fragariae (0.6-4.2%) overlap the divergence (3.6%) within C. gloeosporioides. It is suggested that C. fuscum as well as C. kahawae and C. fragariae fall within the group species C. gloeosporioides. ITS 1 data enabled clear distinction (7.1%) of Colletotrichum isolates from maize and sorghum into C. graminicola and C. sublineolum, respectively (group 2). Species such as C. acutatum, C. coccodes, C. dematium, and C. trichellum can be clearly distinguished based on ITS 1 sequence divergence, but C. destructivum cannot be confidently separated (98% homology) from C. linicola. Colletotrichum dematium f. truncatum is distinct (12.9%) from C. dematium and should probably be called C. truncatum.

A PCR primer-specific to Cylindrocarpon heteronema for detection of the pathogen in apple wood.

An oligonucleotide primer (ChInt) was synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Cylindrocarpon heteronema. PCR with primers ChInt and ITS4 (from a conserved sequence of the rDNA) amplified a 470-bp fragment from several isolates of C. heteronema but not from various apple wood saprophytes. Amplification of this fragment was achieved from 1-2 pg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from cankered wood but only after impurities were removed from the DNA on a Qiagen tip-5 column. Southern hybridization analysis confirmed the 470-bp fragment from C. heteronema DNA and cankered wood to be identical.

Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR.

An oligonucleotide primer (CgInt), synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Collectotrichum gloeosporioides was used for PCR with primer ITS4 (from a conserved sequence of the rDNA) to amplify a 450-bp fragment from the 25 C. gloeosporioides isolates tested. This specific fragment was amplified from as little as 10 fg of fungal DNA. A similar sized fragment was amplified from DNA extracted from C. gloeosporioides-infected tomato tissue. RAPD analysis divided 39 C. gloeosporioides isolates into more than 12 groups linked to host source and geographic origin. Based on the results obtained, the potential of PCR for detection and differentiation of C. gloeosporioides is discussed.

Antagonistic potential of Gliocladium virens and Trichoderma longibrachiatum to phytopathogenic fungi.

Three isolates of Gliocladium virens (G1, G2 and G3) and two of Trichoderma longibrachiatum (T1 and T2) were screened against isolates of three soilborne plant pathogens namely Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum. G. virens exhibited stronger hyperparasitism and wider biological spectrum than T. longibrachiatum. Further, similarities as well as variation was observed in the ability of the various isolates to invade the test pathogens in dual culture. For the hyperparasites, acidic pH range (5.0 to 5.5) favoured both growth and spore germination. The hyperparasites made direct contact with the pathogens followed by varied modes of attack invariably leading to cell disruption. Antagonists, G1 and G3 revealed strong antibiosis while T2 showed moderate effect. All the isolates produced enhanced levels of lytic enzymes adaptively and there were marked differences among them. However, no correlation was observed between these attributes and the hyperparasitic potential of the various isolates in dual culture. The relevance and the role of enzymes and toxic metabolite(s) in the antagonism of G. virens and T. longibrachiatum to these pathogens are discussed.