The Biocontrol Agent Phlebiopsis gigantea: Efficacy and Impacts on the Stump Bacterial Biota and Conifer tree Defences

Phlebiopsis gigantea has been for a long time known as a strong competitor against Heterobasidion annosum and intensively applied as a biological control agent on stump surfaces of Picea abies in Fennoscandia. However, the mechanism underlying its antagonistic activity is still unknown. A primary concern is the possible impact of P. gigantea treatment on resident non-target microbial biota of conifer stumps. Additional risk factor is the potential of P. gigantea to acquire a necrotrophic habit through adaptation to living wood tissues. This study focused on the differential screening of several P. gigantea isolates from diverse geographical sources as well as the use of breeding approach to enhance the biocontrol efficacy against H. annosum infection. The results showed a significant positive correlation between growth rate in wood and high biocontrol efficacy. Furthermore, with aid of breeding approach, several progeny strains were obtained that had better growth rate and control efficacy than parental isolates. To address the issue of the potential of P. gigantea to acquire necrotrophic capability, a combination of histochemical, molecular and transcript profiling (454 sequencing) were used to investigate the interactions between these two fungi and ten year old P. sylvestris seedlings. The results revealed that both P. gigantea and H. annosum provoked strong necrotic lesions, but after prolonged incubation, P. gigantea lesions shrank and ceased to expand further. Tree seedlings pre-treated with P. gigantea further restricted H. annosum-induced necrosis and had elevated transcript levels of genes important for lignification, cell death regulation and jasmonic acid signalling. These suggest that induced localized resistance is a contributory factor for the biocontrol efficacy of P.gigantea, and it has a comparatively limited necrotrophic capability than H. annosum. Finally, to investigate the potential impact of P. gigantea on the stump bacterial biota, 16S rDNA isolated from tissue samples from stumps of P. abies after 1-, 6- and 13-year post treatment was sequenced using bar-coded 454 Titanium pyrosequencing. Proteobacteria were found to be the most abundant at the initial stages of stump decay but were selectively replaced by Acidobacteria at advanced stages of the decay. Moreover, P. gigantea treatment significantly decreased the bacterial richness at initial decay stage in the stumps. Over time, the bacterial community in the stumps gradually recovered and the negative effects of P. gigantea was attenuated.

Bovine mastitis caused by coagulase-negative staphylococci : host response and bacterial factors

Coagulase-negative staphylococci (CNS) are the most common bacteria isolated in bovine subclinical mastitis in many countries, and also a frequent cause of clinical mastitis. The most common species isolated are Staphylococcus (S) chromogenes, S. simulans, S. epidermidis, and S. xylosus. One half of the intramammary infections (IMI) caused by CNS persist in the udder. The pathogenesis of IMI caused by CNS is poorly understood. This dissertation focuses on host response in experimental intramammary infection induced by S. chromogenes, S. epidermidis and S. simulans. Model for a mild experimental CNS infection was developed with S. chromogenes (study I). All cows were infected and most developed subclinical mastitis. In study II the innate immune response to S. epidermidis and S. simulans IMI was compared in eight cows using a crossover design. A larger dose of bacteria was used to induce clinical mastitis. All cows became infected and showed mild to moderate clinical signs of mastitis. S. simulans caused a slightly stronger innate immune response than S. epidermidis, with significantly higher concentrations of the interleukins IL-1beta and IL-8 in the milk. The spontaneous elimination rate of the 16 IMIs was 31%, with no difference between species. No significant differences were recorded between infections eliminated spontaneously or remaining persistent, although the response was stronger in IMIs eliminated spontaneously, except the concentration of TNF-α, which remained elevated in persistent infections. Lactoferrin (Lf) is a component of the humoral defence of the host and is present at low concentrations in the milk. The concentration of Lf in milk is high during the dry period, in colostrum, and in mastitic milk. The effect of an inherent, high concentration of Lf in the milk on experimental IMI induced with S. chromogenes was studied in transgenic cows that expressed recombinant human Lf in their milk. Human Lf did not prevent S. chromogenes IMI, but the host response was milder in transgenic cows than in normal cows, and the former eliminated infection faster. Biofilm production has been suggested to promote persistence of IMI. Phenotypic biofilm formation and slime producing ability of CNS isolates from bovine mastitis was investigated in vitro. One-third of mastitis isolates produced biofilm. Slime production was less frequent for isolates of the most common mastitis causing species S. chromogenes and S. simulans compared with S. epidermidis. No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was associated with persistent infections, but only 8% of isolates produced slime.