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Timothy is a forage mainly grown in Min County, Gansu Province, China. In 2021, a leaf spot disease outbreak on timothy grass occurred in Min County, adversely affecting its growth and productivity. Therefore, this study investigated the leaf spot disease incidence in Min County, morphologically and molecularly characterized the disease-causing pathogen, and assessed its effects on the growth, photosynthesis, and biomass of timothy seedlings re-inoculated with the isolated pathogen. In the field, the disease incidence on plants and leaves was 100 and 85%, respectively. Morphologically, young lesions were ellipsoidal-fusiform with dark purple margins and an off-white center, while the mature lesions were eye-shaped spots with a light brown center and dark purple edges. Molecular characterization identified the pathogen as Cladosporium phlei causing Cladosporium eyespot disease. The net photosynthetic rate, transpiration rate, fresh shoot weight, and dry shoot weight of timothy seedlings 14 days after inoculation with the pathogen were decreased by 29.77, 56, 45.45, and 46.42%, respectively, implying that Cladosporium eyespot disease is an important timothy grass disease in Min County. Therefore, developing an integrated control strategy is urgent to lessen the economic loss., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The occurrence of interaction between insects and fungi is interesting from an ecological point of view, particularly when these interactions involve insect pests and plant pathogens within an agroecosystem. In this study, we aimed to perform an accurate analysis on the fungal microbiota associated to Bactrocera oleae (Rossi) through a metabarcoding approach based on 454 pyrosequencing. From this analysis, we retrieved 43,549 reads that clustered into 128 operational taxonomic units (OTUs), of which 29 resulted in the "core" associate fungi of B. oleae. This fungal community was mainly represented by sooty mould fungi, such as Cladosporium spp., Alternaria spp. and Aureobasidium spp., by plant pathogens like Colletotrichum spp. and Pseudocercospora spp., along with several other less abundant taxa whose ecology is unclear in most of the cases. Our findings lead to new insights into the microbial ecology of this specific ecological niche, enabling the understanding of a complex network of interactions within the olive agroecosystem.

A gene has been identified in tomato, which confers resistance to Cladosporium fulvum through recognition of the pathogenicity factor ECP2. Segregation analysis of F2 and F3 populations showed monogenic dominant inheritance, as for previously reported Cf resistances. The gene has been designated Cf-ECP2. Using several mapping populations, Cf-ECP2 was accurately mapped on chromosome 1, 7.7 cM proximal to TG236 and 6.0 cM distal to TG184. Although Cf-ECP2 is linked to Cf-4, it is not located in the Hcr9 cluster "Milky Way". Therefore, Cf-ECP2 is the first functional Cf homologue on chromosome 1 that does not belong to this Hcr9 cluster. No recombination events between Cf-ECP2 and CT116 have been observed in three populations tested, representing 282 individuals. The low value for the physical distance per cM around CT116 reported previously and the high probability that Cf-ECP2 is also a member of a Hcr9 cluster will facilitate cloning of the locus.

The avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is highly induced during infection of tomato plants. Expression of the Avr9 gene can also be induced in vitro when cells are grown on synthetic liquid medium containing little or no nitrogen. The Avr9 promoter contains six copies of the sequence TAGATA and six additional copies of the core sequence GATA within 0.4 kb upstream of the translation start site. In the filamentous fungi Aspergillus nidulans and Neurospora crassa, these promoter sequences have been identified as the binding sites for a wide-domain GATA-type regulator (AREA in A. nidulans and NIT2 in N. crassa) involved in nitrogen utilization. Quantification of GUS activity of A. nidulans transformants containing a single copy of the fully active Avr9 promoter-uidA (GUS) reporter gene fusion in different areA backgrounds, following starvation for nitrogen, showed that induction of the Avr9 promoter is regulated similarly in A. nidulans and C. fulvum. This suggests that AREA can regulate the Avr9 promoter and that C. fulvum contains an AREA-like regulator that can bind to these specific sequence motifs. Comparison of the induction profiles of Avr9 and niaD showed that Avr9 expression is independent of NIRA, as is niaD expression upon nitrogen starvation. Studies with Avr9 promoter-uidA fusions in which all or most of these sequences had been deleted, showed that Avr9 promoter activity is dependent on the presence of these specific cis-regulatory elements, suggesting that they do indeed function in transcriptional regulation of the Avr9 gene.

Five different hydrophobin-encoding cDNA clones from Cladosporium fulvum were isolated from cDNA libraries, made from nutrient-depleted mycelium. One cDNA clone was identical to the previously isolated hydrophobin HCf-1. The other clones were named HCf-2, -3, -4 and -5. HCf-1, -2, -3 and -4 show a high degree of identity, and are predicted to encode class I hydrophobins. HCf-5 encodes a class II hydrophobin. The expression patterns of these hydrophobins at various stages of development, and in liquid media lacking either carbon or nitrogen, or both, showed clear differences. All hydrophobins were more strongly expressed during sporulation than before, with HCf-4 and -5 showing the highest increase. Expression of HCf genes in infected plants was also higher at 16 days than at 10 days after infection. The expression of HCf-5 in sporulating mycelium was much lower in planta than in vitro. All HCf genes were upregulated under conditions of nutrient deprivation. HCf-1, -2, -3 and -4 showed highest levels of transcription in medium lacking both carbon and nitrogen. Expression of HCf-5 was highest in medium lacking nitrogen but containing carbon. HCf-1 was generally the most abundant hydrophobin. The introduction of multiple copies of HCf-1, which caused co-suppression of the endogenous HCf-1 gene, was shown to affect the expression of HCf-2, -3 and -4 also. Expression of HCf-4 was suppressed, but expression of HCf-2 and -3 was upregulated. Expression of HCf-5 was not changed.

Transformation of Cladosporium fulvum with DNA containing a truncated copy of the hydrophobin gene HCf-1 causes co-suppression of hydrophobin synthesis in 30% of the transformants. The co-suppressed isolates have a hydrophilic phenotype, lower levels of HCf-1 mRNA than wild type and contain multiple copies of the plasmid integrated as tandem repeats at ectopic sites in the genome. Gene silencing is not associated with DNA cytosine methylation. Nuclear run-off experiments reveal that transcription rate of HCf-1 in the co-suppressed isolates is higher than in the untransformed strains, suggesting that silencing acts at the post-transcriptional level. We show, for the first time in fungi, that co-suppression is correlated with the presence of antisense RNA, and that this is synthesised on a DNA template. Derivatives showing reversion to the wild-type phenotype and restoration of HCf-1 gene expression were also observed. Reversion is associated with loss of some copies of the transgene. We propose that co-suppression is due to ectopic integration of the transgene next to promoters which initiate transcription to form antisense RNA and that this in turn determines down-regulation of HCf-1.

A retrotransposon from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva) has been isolated and characterised. It is 6968 bp in length and bounded by identical long terminal repeats of 427 bp; 5 bp target-site duplications were found. Putative first- and second-strand primer binding sites were identified. Three long open reading frames (ORFs) are predicted from the sequence. The first has homology to retroviral gag genes. The second includes sequences homologous to protease, reverse transcriptase, RNAse H and integrase, in that order. Sequence comparisons of the predicted ORFs indicate that this element is closely related to the gypsy class of LTR retrotransposons. Races of the pathogen exhibit polymorphisms in their complement of at least 25 copies of the sequence. Virus-like particles which co-sediment with reverse transcriptase activity were observed in homogenates of the fungus. This is the first report of an LTR retrotransposon in a filamentous fungus.

Methods are described for the electrophoretic separation of chromosome-sized DNA molecules from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva). Using a hexagonal electrode array and switching times of 75 min at 45 V for 14 days, nine bands could be resolved. By comparison with co-electrophoresed Aspergillus nidulans chromosomal DNA (which was resolved into seven bands), the sizes of the C. fulvum bands are estimated to be between 1.9 Mb and 5.4 Mb. The two largest bands are believed to be doublets, giving a minimum genome size of 44 Mb. Cloned probes for the ribosomal DNA repeat, an anonymous single copy fragment and a newly discovered retrotransposon were hybridized to blots of the pulsed field gels, demonstrating the use of this technique for genomic mapping. Most strains of C. fulvum had an identical pattern of bands. Two strains exhibited two polymorphisms which could be due to a translocation.