Polyketide synthases of Diaporthe helianthi and involvement of DhPKS1 in virulence on sunflower.

BACKGROUND: The early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D. helianthi population. RESULTS: In order to investigate the role of polyketide synthases in D. helianthi 7/96, a draft genome of this isolate was examined. We were able to find and phylogenetically analyse 40 genes putatively coding for polyketide synthases (PKSs). Analysis of their domains revealed that most PKS genes of D. helianthi are reducing PKSs, whereas only eight lacked reducing domains. Most of the identified PKSs have orthologs shown to be virulence factors or genetic determinants for toxin production in other pathogenic fungi. One of the genes (DhPKS1) corresponded to the previously cloned D. helianthi lovB gene fragment and clustered with a nonribosomal peptide synthetase (NRPS) -PKS hybrid/lovastatin nonaketide like A. nidulans LovB. We used DhPKS1 as a case study and carried out its disruption through Agrobacterium-mediated transformation in the isolate 7/96. D. helianthi DhPKS1 deleted mutants were less virulent to sunflower compared to the wild type, indicating a role for this gene in the pathogenesis of the fungus. CONCLUSION: The PKS sequences analysed and reported here constitute a new genomic resource that will be useful for further research on the biology, ecology and evolution of D. helianthi and generally of fungal plant pathogens.

The Constitutive Endopolygalacturonase TvPG2 Regulates the Induction of Plant Systemic Resistance by Trichoderma virens.

Trichoderma spp. are opportunistic fungi some of which are commonly present in the rhizosphere. Several species, such as T. virens, are also efficient biocontrol agents against phytopathogenic fungi and exert beneficial effects on plants. These effects are the consequence of interactions between Trichoderma and plant roots, which trigger enhanced plant growth and induce plant resistance. We have previously shown that T. virens I10 expresses two endopolygalacturonase genes, tvpg1 and tvpg2, during the interaction with plant roots; tvpg1 is inducible while tvpg2 is constitutively transcribed. Using the same system, the tomato polygalacturonase-inhibitor gene Lepgip1 was induced at the same time as tvpg1. Here we show by gene disruption that TvPG2 performs a regulatory role on the inducible tvpg1 gene and in triggering the plant immune response. A tvpg2-knockout strain fails to transcribe the inducible tvpg1 gene in neither in vitro in inducing media containing pectin or plant cell walls, nor during the in vivo interaction with tomato roots. Likewise, the in vivo induction of Lepgip1 does not occur, and its defense against the pathogen Botrytis cinerea is significantly reduced. Our data prove the importance of a T. virens constitutively produced endopolygalacturonase in eliciting plant induced systemic resistance against pathogenic fungi.

Use of a non-homologous end-joining-deficient strain (delta-ku70) of the biocontrol fungus Trichoderma virens to investigate the function of the laccase gene lcc1 in sclerotia degradation.

The aim of this study was to apply a generated Δtku70 strain with increased homologous recombination efficiency from the mycoparasitic fungus Trichoderma virens for studying the involvement of laccases in the degradation of sclerotia of plant pathogenic fungi. Inactivation of the non-homologous end-joining pathway has become a successful tool in filamentous fungi to overcome poor targeting efficiencies for genetic engineering. Here, we applied this principle to the biocontrol fungus T. virens, strain I10, by deleting its tku70 gene. This strain was subsequently used to delete the laccase gene lcc1, which we found to be expressed after interaction of T. virens with sclerotia of the plant pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Lcc1 was strongly upregulated at early colonization of B. cinerea sclerotia and steadily induced during colonization of S. sclerotiorum sclerotia. The Δtku70Δlcc1 mutant was altered in its ability to degrade the sclerotia of B. cinerea and S. sclerotiorum. Interestingly, while the decaying ability for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum sclerotia was even enhanced, suggesting the operation of different mechanisms in the mycoparasitism of these two types of sclerotia by the laccase LCC1.

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain.

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.

Plasmid distribution in European Diaporthe helianthi isolates.

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe, which has been sporadically recorded in Italy. A collection of 26 Diaporthe helianthi isolates deriving from different geographic origins was analysed in order to determine the presence of extra-chromosomal genetic determinants and their molecular diversity. Extra-chromosomal bands in total genomic DNAs were identified in every French and the Yugoslavian isolate and in only one Italian isolate, while no Romanian and Argentinean isolates resulted to host any plasmids. When tested for their chemicophysical nature, they were recognised as linear plasmids sized about 2.3 Kb. A more detailed analysis was performed on a plasmid purified from a French isolate (plasmid F). Its intracellular localisation resulted as mitochondrial. Plasmid F was also exploited as a probe in Southern hybridisation experiments, in which it recognised only plasmids present in the genomes of French and Yugoslavian isolates (countries were the disease has a heavy incidence) indicating a strong correlation to geographic origin. An RFLP hybridisation analysis performed on genomic DNAs revealed a homogeneous restriction pattern in all French and Yugoslavian isolates, suggesting molecular homology among plasmids present in those isolates.

A coding region in Diaporthe helianthi reveals genetic variability among isolates of different geographic origin.

A set of Diaporthe helianthi isolates collected in different geographic areas was studied in order to examine whether different genetic biotypes could be responsible for epidemiological differences shown by sunflower stem canker. D. helianthi causes serious losses in France and in former Yugoslavia, while the pathogen is only sporadically recorded in Italy in spite of conducive pedoclimatic conditions. Variability of a D. helianthi coding genomic region, evaluated by means of polymerase chain reaction (PCR), Southern blot hybridisation and restriction fragments length polymorphism (RFLP), showed a conserved homogeneous pattern shared by French and Yugoslavian isolates compared with the heterogeneous pattern of Italian isolates. These results are consistent with other investigations (IGS and ITS region variability) performed on the same set of isolates, allowing a correlation between D. helianthi biotypes, their geographic origin and sunflower stem canker epidemiology.