Effects of a prothioconazole- and tebuconazole-based fungicide on the yield, silage characteristics, and fungal mycobiota of corn harvested and conserved as whole-crop and high-moisture ear silages.

AIMS: To analyze the effect of a prothioconazole- and tebuconazole-based fungicide on the yield and silage characteristics of whole-crop corn (WCC) and high-moisture ear corn (HMC) silages and on the fungal community dynamics from the harvest to aerobic exposure. METHODS AND RESULTS: Corn were untreated (NT) or treated (T) with a prothioconazole- and tebuconazole-based fungicide and harvested as WCC and HMC. Silages were conserved for 60 and 160 d and subjected to an aerobic stability test. The fungicide increased the yield per hectare however, it did not affect the main nutritional characteristics of WCC or HMC. The main chemical, fermentative and microbial characteristics, dry matter (DM) losses and aerobic stability were mainly affected by the conservation time, regardless of the treatment. Fusarium, Alternaria, Aspergillus, and Penicillium genera were identified as dominant before ensiling, but Aspergillus and Penicillium became dominant after silo opening and aerobic exposure. Yeast population during ensiling and aerobic deterioration resulted in a simplification, with Pichia and Kazachstania genera being dominant. CONCLUSIONS: The application of fungicide improved the DM, starch, and net energy for lactation (NEL) yield per hectare but had no consistent effect on the microbial and fermentative silage quality and aerobic stability.

Temperature during conservation in laboratory silos affects fermentation profile and aerobic stability of corn silage treated with Lactobacillus buchneri, Lactobacillus hilgardii, and their combination.

The environment temperature and its effect on the temperature of silage is very important for the fermentation and subsequent quality of a silage. Obligate heterofermentative lactic acid bacteria (LAB) inocula, because of their ability to inhibit yeasts, have been developed to prevent the aerobic deterioration of silages. The temperature during silage conservation may also play an important role in the fermentation profile of silages. This study has evaluated the effect of temperature, during the conservation of whole crop corn silage, untreated or treated with different LAB inocula, on the fermentation profile and on the aerobic stability of the silage. Corn was harvested at 42% dry matter and either not treated (control) or treated with Lactobacillus buchneri NCIMB 40788 (LB) at 300,000 cfu/g fresh matter (FM); Lactobacillus hilgardii CNCM I-4785 at 150,000 cfu/g FM (LH150); L. hilgardii CNCM I-4785 at 300,000 cfu/g FM (LH300); or LB+LH at 150,000 cfu/g FM each. In an attempt to experimentally simulate temperature fluctuations in the mass or at the periphery of a silage bunker, corn was conserved in laboratory silos at a constant temperature (20 ± 1°C; MASS) or at lower and variable outdoor temperatures (PERIPH; ranging from 0.5 to 19°C), and the silos were opened after 15, 30, and 100 d of conservation. Lactic acid, acetic acid, and ethanol contents increased in all the silages over the conservation period. The lactic acid content was higher (+10%) in the silages kept at a constant temperature than those conserved at the lower and variable outdoor temperatures. The acetic acid was higher in the treated silages than in the control ones conserved at a constant temperature for 100 d. Moreover, 1,2-propanediol was only detected in the treated silages after at least 30 d at a constant temperature, whereas only traces were detected in the LB+LH treatment for the other temperature conditions. The yeast count decreased during conservation at a slower rate in PERIPH than in MASS and on average reached 2.96 and 4.71 log cfu/g for MASS and PERIPH, respectively, after 100 d of conservation. The highest aerobic stability values were observed for LH300 (191 h) in the MASS silage after 100 d of conservation, whereas the highest aerobic stability was observed in LB+LH (150 h) in the PERIPH silages. After 7 d of air exposure, a pH higher than 4.5 and a higher yeast than 8.0 log cfu/g were detected in all the silages opened after 15 and 30 d of conservation. A pH value close to that of silo opening was detected in the LB, LH150, and LH300 silages conserved under MASS conditions after 100 d, whereas LB+LH was the most effective under PERIPH conditions. The temperature and its fluctuation during conservation of silage in laboratory silos influenced the fermentation, which in turn had an effect on the quality of silage and on the extent of the effect of LAB inocula.

Increase in aflatoxins due to Aspergillus section Flavi multiplication during the aerobic deterioration of corn silage treated with different bacteria inocula.

The growth of Aspergillus flavus and the production of aflatoxins (AF) during the aerobic deterioration of corn silage represent a problem for animal and human health. This experiment was conducted to evaluate whether the presence of A. flavus and AF production originate from the field or additional AF are produced during the fermentation phase or during aerobic deterioration of corn silage. The trial was carried out in northern Italy on corn at a dry matter (DM) level of 34%. The fresh herbage was either not treated (C) or treated with a Lactobacillus buchneri (LB) NCIMB 40788 [(at 3 × 105 cfu/g of fresh matter (FM)], Lactobacillus hilgardii (LH) CNCM I-4785 (at 3 × 105 cfu/g of FM), or their combination (LB+LH; at 1.5 × 105 cfu/g of FM of each strain) ensiled in 20-L silos and opened after 250 d of ensiling. After silo opening, the aerobic stability was evaluated and samples were taken after 7 and 14 d of air exposure. The pre-ensiled material, the silages at silo opening, and the aerobically exposed silages were analyzed for DM content, fermentative profiles, microbial count, nutritive characteristics, DM losses, and AFB1, AFB2, AFG1, and AFG2 contents. Furthermore, a subsample of colonies with macromorphological features of Aspergillus section Flavi was selected for AF gene pattern characterization and in vitro AF production. The presence of A. flavus was below the detection limit (<1.00 log10 cfu/g) in the fresh forage before ensiling, whereas it was found in 1 out of 16 silage samples at silo opening at a level of 1.24 log10 cfu/g. The AF were found in both the fresh forage and at opening in all the samples, with a predominance of AFB2 (mean value of 1.71 µg/kg of DM). The inoculation of lactic acid bacteria determined a reduction in the lactic-to-acetic ratio compared with the control. A larger amount of acetic acid resulted in a lower yeast count and higher aerobic stability in the treated silages than in the control ones. At the beginning of aerobic deterioration, the yeasts increased to over 5 log10 cfu/g, whereas the molds were close to the value observed at silo opening. When the inhibiting conditions were depleted (pH and temperature higher than 5 and 35°C, respectively), both the total molds and A. flavus reached higher values than 8.00 and 4.00 log10 cfu/g, respectively, thus determining the ex novo production of AFB1 during aerobic deterioration, regardless of treatments. The analysis of gene pattern showed that 64% of the selected colonies of A. flavus showed the presence of all 4 AF gene patterns, and 43% of the selected colonies were able to produce AF in vitro. During air exposure, after 1,000°C·h have been cumulated, starch content decreased (below 10% DM) and concentration of neutral detergent fiber, acid detergent fiber, hemicelluloses, crude protein, and ash increased. The inoculation with LB and LB+LH increased the aerobic stability of the silages and delayed the onset of aerobic microbial degradation, which in turn indirectly reduced the risk of A. flavus outgrowth and AFB1 production after silage opening.

Effects of conservation period and Lactobacillus hilgardii inoculum on the fermentation profile and aerobic stability of whole corn and sorghum silages.

BACKGROUND: Lactic acid bacteria inocula have been developed over the years to improve the aerobic stability of silages. The aims of the study were to evaluate the effect of various conservation periods and the use of Lactobacillus hilgardii inoculum on aerobic stability, fermentative profile and microbial population of corn and sorghum silages. Trials were carried out on two corns and one sorghum crops. The crops were untreated or treated with L. buchneri (LB, application rate 300 000 cfu g-1 FM), L. hilgardii (LH, application rate 300 000 cfu g-1 FM) and a combination (LB + LH, application rate 150 000 cfu g-1 FM each). Silos were opened after 15, 30, 100 and 250 days of conservation, and the silages were analysed for fermentative profile, microbial count and aerobic stability. RESULTS: During conservation, the inocula influenced the fermentation profile. The use of LH increased the aerobic stability at 15 and 30 days in one out of three trials, while after 100 and 250 days, the presence of LB alone or with LH led to greater stability. In all the trials, the acetic acid content increased, the yeast count decreased and the aerobic stability increased during the conservation period. CONCLUSIONS: This study has shown that a long period in complete anaerobiosis reduced yeast count and improved aerobic stability in all silages. The addition of LB was confirmed to be a good option for increasing aerobic stability of silages, whereas the effect of LH alone or in combination with LB on aerobic stability was not consistent between trials. © 2018 Society of Chemical Industry.

Augmented visualization by computer vision and chromatographic fingerprinting on comprehensive two-dimensional gas chromatographic patterns: Unraveling diagnostic signatures in food volatilome.

Computer Vision is an approach of Artificial Intelligence (AI) that conceptually enables "computers and systems to derive useful information from digital images" giving access to higher-level information and "take actions or make recommendations based on that information". Comprehensive two-dimensional chromatography gives access to highly detailed, accurate, yet unstructured information on the sample's chemical composition, and makes it possible to exploit the AI concepts at the data processing level (e.g., by Computer Vision) to rationalize raw data explorations. The goal is the understanding of the biological phenomena interrelated to a specific/diagnostic chemical signature. This study introduces a novel workflow for Computer Vision based on pattern recognition algorithms (i.e., combined untargeted and targeted UT fingerprinting) which includes the generation of composite Class Images for representative samples' classes, their effective re-alignment and registration against a comprehensive feature template followed by Augmented Visualization by comparative visual analysis. As an illustrative application, a sample set originated from a Research Project on artisanal butter (from raw sweet cream to ripened butter) is explored, capturing the evolution of volatile components along the production chain and the impact of different microbial cultures on the finished product volatilome. The workflow has significant advantages compared to the classical one-step pairwise comparison process given the ability to realign and pairwise compare both targeted and untargeted chromatographic features belonging to Class Images resembling chemical patterns from many different samples with intrinsic biological variability.

Effect of Inoculation with Lentilactobacillus buchneri and Lacticaseibacillus paracasei on the Maize Silage Volatilome: The Advantages of Advanced 2D-Chromatographic Fingerprinting Approaches.

In this study, the complex volatilome of maize silage samples conserved for 229 d, inoculated with Lentilactobacillus buchneri (Lbuc) and Lacticaseibacillus paracasei (Lpar), is explored by means of advanced fingerprinting methodologies based on comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry. The combined untargeted and targeted (UT) fingerprinting strategy covers 452 features, 269 of which were putatively identified and assigned within their characteristic classes. The high amounts of short-chain free fatty acids and alcohols were produced by fermentation and led to a large number of esters. The impact of Lbuc fermentation was not clearly distinguishable from the control samples; however, Lpar had a strong and distinctive signature that was dominated by propionic acid and 1-propanol characteristic volatiles. The approach provides a better understanding of silage stabilization mechanisms against the degradative action of yeasts and molds during the exposure of silage to air.

Lentilactobacillus hilgardii Inoculum, Dry Matter Contents at Harvest and Length of Conservation Affect Fermentation Characteristics and Aerobic Stability of Corn Silage.

Heterofermentative Lentilactobacillus hilgardii isolated from sugarcane silage, has recently been proposed as a silage inoculant to increase aerobic stability. Various conditions can influence the activity of LAB and their ability to alter silage quality (e.g., DM content and length of conservation). The aim of this study has been to evaluate the effect of L. hilgardii on the fermentation quality and aerobic stability of whole crop corn silage with different DM contents (from 26 to 45%), conserved for various conservation lengths (13-272 days). The silages were analyzed for their DM content, pH, fermentative profile, microbial count, and aerobic stability. L. hilgardii showed a positive effect on improving the aerobic stability of silages, due its ability to produce acetic acid, and reduced the yeast count. The acetic acid content increased as the conservation period increased and decreased as the DM content increased. The yeast count was reduced during conservation in a DM dependent manner and the inoculation with LH determined a reduction in the count of 0.48 log cfu/g. The aerobic stability increased as the conservation period increased, and the treatment with LH on average increased the aerobic stability by 19 h. The results of this experiment suggest that higher aerobic stability could be achieved in corn silages by ensiling at medium or low DM contents, or by increasing the length of conservation if a higher DM content at ensiling is needed. The inoculation with LH helps to improve the aerobic stability of corn silages by reducing the yeast count.

Testing selectivity of bacterial and fungal culture media compared to original silage samples using next generation sequencing.

Plate counts using selective culture media is still the most frequently used method for the enumeration of the different microbial groups that colonize silage, including lactic acid bacteria, yeasts and molds. Since different culture media have specific composition, they may allow the growth of specific populations. To date, no study has used next generation sequencing technology to compare the selective capacity of these different culture media although this approach could provide comprehensive insight into the relevance of using one culture medium over another. Sequencing of the 16S rDNA and ITS amplicon were performed to compare the selectivity of different culture media used in silage microbiology. Corn silage, grass-alfalfa silage and total mixed ration extracts were plated on five selective media for lactic acid bacteria, incubated under aerobic and anaerobic conditions, and on eight selective media for yeast and molds to compare their selectivity. Ensiling provided a pre-selection environment for specific microorganisms over forage and reduced the number of observed OTUs: only 12 OTUs of bacteria were observed in corn silage sampled in the center of a bunker silo, while the mean number of OTUs identified in samples taken closer to the side of the silo, influenced by higher oxygen and humidity level, increased to 79. Still, MRS and Rogosa plates had less than 12 different OTUs in the center and 24 at the side, mainly Lactobacillaceae, Acetobacteraceae, and Leuconostocaceae. Incubating the plates under anaerobic conditions was selective against Acetobacteraceae. MRS supplemented with acetic acid increased selectivity of lactic acid bacteria. When plated on culture media specific for yeast and molds, from 17 to 68 different OTUs were observed in corn silage. Mixed grass-alfalfa silage and total mixed ration samples usually had more observed OTUs and the diversity profile of the corresponding culture media was similar to that of the original samples. For yeasts and molds, Dichloran Rose Bengal Chloramphenicol Agar revealed a diversity profile close to the that of the corn silage.

Aspergillus fumigatus population dynamics and sensitivity to demethylation inhibitor fungicides in whole-crop corn, high moisture corn and wet grain corn silages.

BACKGROUND: Aspergillus fumigatus, the causal agent of aspergillosis in humans, is commonly present as a saprophyte in various organic substrates, such as spoiled silages. Aspergillosis is generally combated with demethylation inhibitor (DMI) fungicides, but the recent appearance of resistant medical and environmental strains made current treatment strategies less reliable. The goal of this study was to determine the evolution of A. fumigatus populations during the ensiling process of whole-crop corn, high moisture corn and wet grain corn, and to monitor the sensitivity of isolates from treated and untreated fields to one medical and one agricultural DMI fungicide. RESULTS: A. fumigatus was isolated from fresh forage at harvest at rather low concentrations (102 cfu g-1 ). The low frequency lingered during the silage process (at 60 and 160 days), whereas it significantly increased during air exposure (at 7 and 14 days of air exposure). Field treatment of corn with a mixture of prothioconazole and tebuconazole did not affect the sensitivity of A. fumigatus isolates. One of 29 isolates from the untreated plot was resistant to voriconazole. A unique amino acid substitution (E427K) was detected in the cyp51A gene of 10 of 12 sequenced isolates, but was not associated with DMI resistance. CONCLUSION: A. fumigatus significantly increased during aerobic deterioration of ensilaged corn after silo opening, compared with the low presence in fresh corn and during ensiling. Field treatment of corn with DMI fungicides did not affect the sensitivity of A. fumigatus isolates collected from fresh and ensiled corn. © 2019 Society of Chemical Industry.