Design, implementation and interpretation of in vitro batchculture experiments to assess enteric methane mitigation in ruminants—a review

In vitro fermentation techniques (IVFT) have been widely used to evaluate the nutritivevalue of feeds for ruminants and in the last decade to assess the effect of different nutritionalstrategies on methane (CH4) production. However, many technical factors may influencethe results obtained. The present review has been prepared by the ‘Global Network’ FACCE-JPI international research consortium to provide a critical evaluation of the main factorsthat need to be considered when designing, conducting and interpreting IVFT experimentsthat investigate nutritional strategies to mitigate CH4emission from ruminants. Given theincreasing and wide-scale use of IVFT, there is a need to critically review reports in the lit-erature and establish what criteria are essential to the establishment and implementationof in vitro techniques. Key aspects considered include: i) donor animal species and numberof animal used, ii) diet fed to donor animals, iii) collection and processing of rumen fluidas inoculum, iv) choice of substrate and incubation buffer, v) incubation procedures andCH4measurements, vi) headspace gas composition and vii) comparability of in vitro andin vivo measurements. Based on an evaluation of experimental evidence, a set of techni-cal recommendations are presented to harmonize IVFT for feed evaluation, assessment ofrumen function and CH4production.

‘Symptomless’ infection by Botrytis cinerea

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection.