Penicillium roqueforti s.l. : growth and roquefortine C production in silages

On Belgian farms, visible fungal growth is regularly encountered in ensiled feed commodities. The toxigenic fungal species Penicillium roqueforti sensu strictu (s.s.) and P. paneum, designated together as P. roqueforti sensu lato (s.l.) in this dissertation, are the most frequently isolated fungi in silages. Since the inhalation of fungal spores as well as the consumption of mycotoxin contaminated feed comprise serious health risks, it is of the outmost importance to prevent fungal contamination of silages. In this dissertation, general preventory measures are described. The final goal of this PhD research was to contribute to the prevention of specifically P. roqueforti s.l. development in silage. To achieve this goal, multiple in vitro lab experiments and in vivo trials with microsilos have been conducted, evaluating the effect of several abiotic and biotic factors on P. roqueforti s.l. growth and mycotoxin production. Roquefortine C, a mycotoxin that can be produced by both P. roqueforti s.s. and P. paneum, is considered to be an indicator of mycotoxin production by P. roqueforti s.l. in silages. Therefore, this particular mycotoxin has been determined to evaluate mycotoxin production. During the ensiling process, lactic acid bacteria convert sugars to mainly lactic acid, but also some acetic acid, methanol, ethanol, etc. These compounds can be used by P. roqueforti s.l. as a carbon source. Lactic acid as sole carbon source was not very conducive for fungal growth, while acetic acid (inhibiting aerobic deterioration and subsequent fungal development in silages) as sole carbon source facilitated good fungal growth. This illustrates that P. roqueforti s.l. is very well adapted to its silage-habitat, rendering prevention difficult. The bacterium Bacillus velezensis displayed antagonistic properties towards P. roqueforti s.l. in an in vitro experiment: both culture supernatant as cell suspension reduced spore germination and spore survival and inhibited fungal growth, without triggering an increased roquefortine C production. These observations seemed promising towards the capability of B. velezensis to prevent P. roqueforti s.l. development in silages, but the applied cell suspension could not live up to the great expectations regarding antagonism in an in vivo microsilo trial. Future research is required to investigate the potential of B. velezensis as a silage additive to counter P. roqueforti s.l. in silage. Oxygen appears to play a crucial role in the development of P. roqueforti s.l.: in anaerobic conditions, no fungal growth can occur. An in vivo microsilo trial with artificially infected whole-crop maize (@ 1500 conidia per gram fresh matter) desiled after 50, 100 or 150 days demonstrated that at desiling after 50 days some P. roqueforti s.l. propagules (66 per gram verse stof) had survived, while after an ensiled period of 100 days no active P. roqueforti s.l. propagules were detected. This experiment emphasizes the importance of a sufficiently long ensiled period, during which the integrity of the silo coverage needs to be maintained. In order to prevent the development of P. roqueforti s.l. in silages, the strict application of good agricultural practices regarding ensiling and desiling, limiting air ingress into silages, is the key factor to success

The effect of a mixture of Lactobacillus strains on silage quality and nutritive value of grass harvested at four growth stages and ensiled for two periods

The effect of adding an inoculant containing Lactobacillus buchneri, L. plantarum and L. casei to wilted perennial ryegrass, harvested at four growth stages and ensiled for either 60 or 150 d on silage fermentation quality, chemical composition, rumen degradability of neutral detergent fibre (NDF) and organic matter (OM) and in vitro OM digestibility (OMd) was studied. Compared to the control silage, more sugars were fermented to lactic and acetic acid with the inoculant, resulting in a lower pH, less dry matter losses and protein degradation and a better aerobic stability. The effects of the additive on fermentation quality were more pronounced after 150 than after 60 d of ensiling, because the quality of the control silage was worse after the long ensiling period. The treatment lowered NDF content of grass harvested at the first two growth stages by degrading cell walls to complex sugars, but had no effect on NDF degradability of the silage. The inoculant had no effect on rumen OM degradability nor on OMd after the short ensiling period, but increased the rumen OM degradability for the first two growth stages and OMd for all growth stages after the long ensiling period

Survey of fungal diversity in silages supplied to dairy cattle in Belgium over a two-year period

The fungal diversity in silages for dairy cattle feeding has been assessed by purification and identification of 966 isolates collected in silages during the two 2006 and 2007 winter storage/feed-out periods from farms localized in various geographic regions in Belgium. The relevant fungal species in silos were P. paneum and P. roqueforti (18.2 % and 14.5 % of total isolates, respectively). The proportion between the two species varied significantly from 2006 to 2007 (P<0.05) depending on the type of forage crop. The prevalence of P. paneum in Belgium compared to results in other countries is of major importance due to the mycotoxigenic capacity of this species. Information on numerous aspects of silage making process and silo management showed that neither the crop rotation, the date and weather at harvest, the use of additives, the feeding rate, the type and dimension of silo, the covering used nor the forage chopping length at ensiling could be significantly correlated to the fungal species composition isolated in farm silages. There were also no significant relationships between the chemico-fermentative parameters tested (dry matter, pH, NH3 content, concentration of lactic, acetic and butyric acids) and the fungal species composition in silages. The prevalent fungi were tolerant of the wide ranges of conditions found in the farm silages with year-to-year variations. Survey of Fungal Diversity in Silages Supplied to Dairy Cattle in Belgium Over a Two-Year Period. Available from: https://www.researchgate.net/publication/313255127_Survey_of_Fungal_Diversity_in_Silages_Supplied_to_Dairy_Cattle_in_Belgium_Over_a_Two-Year_Period [accessed Jul 24, 2017]

Storage, fertilization and cost properties highlight the potential of dried microbial biomass as organic fertilizer

The transition to sustainable agriculture and horticulture is a societal challenge of global importance. Fertilization with a minimum impact on the environment can facilitate this. Organic fertilizers can play an important role, given their typical release pattern and production through resource recovery. Microbial fertilizers (MFs) constitute an emerging class of organic fertilizers and consist of dried microbial biomass, for instance produced on effluents from the food and beverage industry. In this study, three groups of organisms were tested as MFs: a high-rate consortium aerobic bacteria (CAB), the microalgaArthrospira platensis('Spirulina') and a purple non-sulfur bacterium (PNSB)Rhodobactersp. During storage as dry products, the MFs showed light hygroscopic activity, but the mineral and organic fractions remained stable over a storage period of 91 days. For biological tests, a reference organic fertilizer (ROF) was used as positive control, and a commercial organic growing medium (GM) as substrate. The mineralization patterns without and with plants were similar for all MFs and ROF, with more than 70% of the organic nitrogen mineralized in 77 days. In a first fertilization trial with parsley, all MFs showed equal performance compared to ROF, and the plant fresh weight was even higher with CAB fertilization. CAB was subsequently used in a follow-up trial with petunia and resulted in elevated plant height, comparable chlorophyll content and a higher amount of flowers compared to ROF. Finally, a cost estimation for packed GM with supplemented fertilizer indicated that CAB and a blend of CAB/PNSB (85%/15%) were most cost competitive, with an increase of 6% and 7% in cost compared to ROF. In conclusion, as bio-based fertilizers, MFs have the potential to contribute to sustainable plant nutrition, performing as good as a commercially available organic fertilizer, and to a circular economy

Purple non‐sulphur bacteria and plant production: benefits for fertilization, stress resistance and the environment

Purple non-sulphur bacteria (PNSB) are phototrophic microorganisms, which increasingly gain attention in plant production due to their ability to produce and accumulate high-value compounds that are benefi- cial for plant growth. Remarkable features of PNSB include the accumulation of polyphosphate, the pro- duction of pigments and vitamins and the production of plant growth-promoting substances (PGPSs). Scattered case studies on the application of PNSB for plant cultivation have been reported for decades, yet a comprehensive overview is lacking. This review highlights the potential of using PNSB in plant pro- duction, with emphasis on three key performanceindicators (KPIs): fertilization, resistance to stress (biotic and abiotic) and environmental benefits. PNSB have the potential to enhance plant growth performance, increase the yield and quality of edible plant biomass, boost the resistance to environmental stresses, bioremediate heavy metals and mitigate greenhouse gas emissions. Here, the mechanisms responsible for these attributes are discussed. A dis- tinction is made between the use of living and dead PNSB cells, where critical interpretation of existing literature revealed the better performance of living cells. Finally, this review presents research gaps that remain yet to be elucidated and proposes a roadmap for future research and implementation paving the way for a more sustainable crop production

The effect of Lactobacillus buchneri inoculation on the aerobic stability and fermentation characteristics of alfalfa-ryegrass, red clover and maize silage

Aerobic spoilage of silages occurs frequently and is undesirable because it reduces both its nutritive and hygienic quality. Silage inoculants containing heterofermentative lactic acid bacteria, like Lactobacillus buchneri, have already been proven to improve aerobic stability by augmented production of acetic acid, which inhibits yeasts. In this study, the effect of L. buchneri on fermentation characteristics and aerobic stability of alfalfa-ryegrass silage, red clover silage and maize silage was assessed using microsilos. Two dosages, 1×105 and 3×105 cfu g-1 of fresh matter, were compared to untreated control silage. Inoculation with L. buchneri clearly altered the fermentation characteristics of alfalfa-ryegrass and red clover silage, resulting in a significantly higher aerobic stability at both dosages. The effects of L. buchneri inoculation on maize silage were less clear, but nevertheless the aerobic stability of maize silage inoculated with 1×105 cfu g-1 of fresh matter was significantly higher compared to the untreated silage

The effect of Lactobacillus buchneri inoculation on the aerobic stability and fermentation characteristics of alfalfa-ryegrass, red clover and maize silage

Aerobic spoilage of silages occurs frequently and is undesirable because it reduces both its nutritive and hygienic quality. Silage inoculants containing heterofermentative lactic acid bacteria, like Lactobacillus buchneri, have already been proven to improve aerobic stability by augmented production of acetic acid, which inhibits yeasts. In this study, the effect of L. buchneri on fermentation characteristics and aerobic stability of alfalfa-ryegrass silage, red clover silage and maize silage was assessed using microsilos. Two dosages, 1x10(5) and 3x10(5) cfu g(-1) of fresh matter, were compared to untreated control silage. Inoculation with L. buchneri clearly altered the fermentation characteristics of alfalfa-ryegrass and red clover silage, resulting in a significantly higher aerobic stability at both dosages. The effects of L. buchneri inoculation on maize silage were less clear, but nevertheless the aerobic stability of maize silage inoculated with 1x10(5) cfu g(-1) of fresh matter was significantly higher compared to the untreated silage

The effect of a mixture of Lactobacillus strains on silage quality and nutritive value of grass harvested at four growth stages and ensiled for two periods

 The effect of adding an inoculant containing Lactobacillus buchneri, L. plantarum and L. casei to wilted perennial ryegrass, harvested at four growth stages and ensiled for either 60 or 150 d on silage fermentation quality, chemical composition, rumen degradability of neutral detergent fibre (NDF) and organic matter (OM) and in vitro OM digestibility (OMd) was studied. Compared to the control silage, more sugars were fermented to lactic and acetic acid with the inoculant, resulting in a lower pH, less dry matter losses and protein degradation and a better aerobic stability. The effects of the additive on fermentation quality were more pronounced after 150 than after 60 d of ensiling, because the quality of the control silage was worse after long ensiling period. The treatment lowered NDF content of grass harvested at the first two growth stages by degrading cell walls to complex sugars, but had no effect on NDF degradability of the silage. The inoculant had no effect on rumen OM degradability nor on OMd after the short ensiling period, but increased the rumen OM degradability for the first two growth stages and OMd for all growth stages after long ensiling period