Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains

A technique to monitor lactic acid bacteria inoculants in silage, based on specific DNA probes, was developed and used to evaluate the colonization properties of two strains of Lactobacillus plantarum and one strain of Pediococcus pentosaceus which were used as maize silage inoculants in farm conditions. The results indicated that these three strains were able to dominate the natural microflora of the silage, representing more than the 95% of the bacterial biomass of the maize silage. These studies indicate that the colony hybridization with specific DNA probes may be an effective method for monitoring bacteria and evaluating the colonization properties of inoculants in maize silage.     

New trends and opportunities in the development and use of inoculants for silage

Abstract: Inoculants are used as silage additives to improve preservation efficiency and to enhance animal performance. In most commercially available inoculants, homofermentative lactic acid bacteria (LAB) have been used because they are fast and efficient producers of lactic acid, improving natural silage fermentation. Specific LAB inuculants may also have beneficial effects on animal performance even if there is no effect on fermentation. However, these types of inoculants are not always advantageous. They do not necessarily prevent sermentation by clostridia in moist silages, and they sometimes impair the aerobic stability of grass and small grain silages. Therefore, new criteria for silage inoculants should be established which consider the specific needs of the crop being ensiled. New approaches which are being taken to develop improved inoculants for silage include the following: (1) using LAB isolates which are more specific to the target crops; (2) inclusion of heterofermentative LAB to produce volatile fatty acids to inhibit yeasts and moulds upon aerobic exposure; (3) inclusion of organisms other than LAB in inoculants to inhibit detrimental microorganisms; (4) selection or engineering of LAB strains to inhibit specific microorganisms; and (5) cloning and expression of genes which would enable selected LAB strains to utilize polysaccharides in crops which are low in soluble carbohydrates. Many of these new strategies for formulating inoculants are being tested, but further research is needed to determine the most successful approaches.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silage

Aims:  To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods.
Methods and Results:  DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony-forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation.
Conclusions:  The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples.
Significance and Impact of the Study:  The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri , a micro-organism used as an inoculant.     

Antibacterial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants

AIMS: To determine antibacterial activity in lactic acid bacteria (LAB) silage inoculants and in wheat and corn silages which were treated with these inoculants. METHODS AND RESULTS: Wheat and two corn silages were prepared in 0.25 l sealed glass jars. Inoculant treatments were prepared for each type of silage with each of 10 LAB silage inoculants at inoculation rate of 10(6) CFU g(-1). Untreated silages served as controls. Antibacterial activity was determined in the inoculants and in their respective silages with Micrococcus luteus and Pseudomonas aeruginosa. Antibacterial activity was detected in nine of the 10 inoculants whereas such activity in the silages varied. Control silages did not have antibacterial activity. CONCLUSIONS: Many LAB silage inoculants have antibacterial activity and in some cases this activity is imparted on inoculated silages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was conducted as part of a broader research objective, which is to find out how LAB silage inoculants enhance ruminant performance. The results of this study indicate that LAB silage inoculants produce antibacterial activity, and therefore, have a potential to inhibit detrimental micro-organisms in the silage or in the rumen.     

Growth and survival of lactic acid bacteria in lucerne silage

A rifampicin-resistant variant of two strains of Lactobacillus plantarum, one strain of Pediococcus acidilactici, and one strain of Enterococcus faecium were used for the experimental production of lucerne silage. Laboratory silage without inoculants served as a control. Counts of total anaerobes, total lactic acid bacteria (LAB), lactobacilli, pediococci, and enterococci were determined on days 14, 21, 30, 49, and 60 of lucerne fermentation. LAB dominated in silage microflora, reaching a percentage between 59 and 95 % of total anaerobes. Lactobacilli were found as a predominant group of LAB during the whole study. Lactobacilli reached numbers 8.74 log CFU/g in treated silage and 8.89 log CFU/g in the control at the first observation. Their counts decreased to 4.23 and 4.92 log CFU/g in treated silage and the control, respectively, on day 63 of fermentation. Similar decreases were observed in all bacterial groups. The treated silage samples possessed lower pH (4.2 vs. 4.5 in control samples) and contained more lactic acid compared to control silage. The identity of re-isolated rifampicin-resistant bacteria with those inoculated to the lucerne was evaluated by fingerprinting techniques. The fingerprint profiles of re-isolated bacteria corresponded to the profiles of strains used for the treatment. It could be concluded that supplemented LAB dominated in laboratory silage and overgrew naturally occurring LAB.     

The survival of silage inoculant lactic acid bacteria in rumen fluid

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.     

玉米秸秆青贮过程中优势细菌多样性分析

通过从发酵0、1、3、5、15、30和60d自然发酵的青贮玉米(CK)和采用菌剂处理的青贮玉米(处理1)上取样,分别进行pH值测定和菌群总DNA的提取。总DNA经纯化后采用引物341F-GC和518R进行细菌16S rDNA V3区PCR扩增,扩增产物进行变性梯度凝胶电泳(DGGE)分离并回收优势条带和变化明显的条带进行测序分析,了解秸秆饲料发酵过程中的优势细菌多样性及动态变化规律,从而为现有青贮接种菌的改良奠定理论基础。研究结果表明:采用菌剂处理的青贮玉米pH值下降更快,其优势细菌种类更丰富,且各优势菌的丰度比对照组中相应菌的丰度更强。     

Identification of Rhizobium spp. in peat-based inoculants by DNA hybridization and PCR and its application in inoculant quality control.

Procedures based on DNA hybridization and PCR were developed for quality control of Rhizobium inoculants. Inoculants for pea and goat's rue were produced by Elomestari Ltd., Juva, Finland, in sterile dry fine peat by the standard procedure used by the company. The inoculants contained Rhizobium galegae HAMBI 1174 and HAMBI 1207 and an R. leguminosarum biovar vicia strain, 16HSA, either solely or in combinations of two or three strains. DNA was isolated from 1-g samples of each peat inoculant and analyzed by nonradioactive DNA-DNA hybridization and by PCR. The hybridization probes were total DNAs from pure cultures of R. galegae HAMBI 1207 and R. leguminosarum biovar viciae 16HSA and a 264-bp strain-specific fragment from the genome of R. galegae HAMBI 1174. The total DNA probes distinguished inoculants containing R. galegae or R. leguminosarum, and the strain-specific probe distinguished inoculants containing R. galegae HAMBI 1174. The hybridization results for R. galegae were verified in a PCR experiment by amplifying an R. galegae species-specific fragment and an R. galegae HAMBI 1174 strain-specific fragment in the same reaction. When suitable probes and primers are available, the methods described here offer promising alternatives for the quality control of peat-based inoculants.     

The influence of bacterial inoculants on the microbial ecology of aerobic spoilage of barley silage.

The aerobic decomposition of barley silage treated with two inoculants (LacA and LacB) containing mixtures of Lactobacillus plantarum and Enterococcus faecium was investigated over a 28-day period. Initially, yeast and bacterial populations were larger in silage inoculated with LacA than in silage treated with LacB or water alone (control). Differences in the succession of yeasts in silage treated with LacA were observed relative to the other two treatments. From silage treatment with LacA, Issatchenkia orientalis was the most prevalent yeast taxon over all of the sample times, and the filamentous fungus Microascus brevicaulis was also frequently isolated at later sample dates (> or = 14 days). In contrast, Saccharomyces exiguus was the most prominent yeast recovered from silage treated with LacB and water alone on days 2 and 4, although it was supplanted by I. orientalis at later sample times. Successional trends of bacteria were similar for all three treatments. Lactobacillus spp. were initially the most prevalent bacteria isolated, followed by Bacillus spp. (primarily Bacillus pumilus). However, the onset of Bacillus spp. prominence was faster in LacA silage, and Klebsiella planticola was frequently recovered at later sample times (> or = 14 days). More filamentous fungi were recovered from LacA silage on media containing carboxylmethylcellulose, pectin, or xylan. The most commonly isolated taxa were Absidia sp., Aspergillus flavus, Aspergillus fumigatus, Byssochlamys nivea, Monascus ruber, Penicillium brevicompactum, Pseudoallescheria boydii, and M. brevicaulis. The results of this study indicated that the two bacterial inoculants incorporated into barley at the time of ensilage affected the microbial ecology of silage decomposition following exposure to air. However, neither of the microbial inoculants effectively delayed aerobic spoilage of barley silage, and the rate of decomposition of silage treated with one of the inoculants (LacA) was actually enhanced.     

Diversity of a stable enrichment culture which is useful for silage inoculant and its succession in alfalfa silage

Alfalfa is a kind of forage that is difficult to ensile for good quality. Therefore, inoculants are always used to enhance the preservation of alfalfa silage. Through continuous restricted subcultivation, a lactic acid bacteria community (Al2) was selected from well-fermented alfalfa silage, which sharply decreased the pH level and produced a large amount of lactic acid. The adding of Al2 to alfalfa at ensiling resulted in a more rapid drop in pH and higher levels of lactic acid, and it also reduced the ammonia-nitrogen content significantly (P < 0.01). Plate isolation, denaturing gradient gel electrophoresis (DGGE) and the construction of a 16S rRNA gene clone library were used to identify the composition diversity of the Al2 community; seven strains were detected in the community, the predominant strain belonging to Lactobacillus plantarum. Samples of alfalfa silages of duration 0, 2, 5, 10, 20 and 30 days were studied with DGGE analysis. The DGGE band patterns of Al2-treated and non inoculated were rather different, and the components of Al2 were the dominant bacteria in Al2-treated silages, especially L. plantarum, while Pediococcus pentosaceaus was predominant in naturally fermented alfalfa silage.     

Immunolocalization of dinitrogenase reductase produced by Klebsiella pneumoniae in association with Zea mays L

The endophytic lifestyle of Klebsiella pneumoniae is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by K. pneumoniae strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for K. pneumoniae and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions.     

Processes of plant colonization by Methylobacterium strains and some bacterial properties

The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacterium are indespensible inhabitants of the plant phyllosphere. Using maize Zea mays as a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied. A marked strain, Methylobacterium mesophilicum APR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves. Colonization of maize leaves by M. mesophilicum APR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface. In this case, the number of PPFMB cells on inoculated leaves increased with plant growth. During seed germination, no colonization of maize leaves with M. mesophilicum cells occurred immediately from the soil inoculated with the marked strain. Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air. PPFMB monocultures were not antagonistic to phytopathogenic bacteria. However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicum or M. extorquens did exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas camprestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense, and Agrobacterium tumifaciens). Neither epiphytic and soil strains of Methylobacterium extorquens, M. organophillum, M. mesophilicum, and M. fujisawaense catalyzed ice nucleation. Hence, they cause no frost injury to plants. Thus, the results indicate that the strains of the genus Methylobacterium can protect plants against adverse environmental factors.     

Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

Aims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real‐time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP‐154 and CFN‐535 in the rhizosphere. Methods and Results: Primers were designed based on strain‐specific SCAR markers and were screened for successful amplification of target strain and absence of cross‐reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real‐time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 × 10³ (for UAP‐154) and 4 × 10⁴ CFU g^?1 (for CFN‐535) in the maize rhizosphere. Inoculant quantification was effective from 10⁴ to 10⁸ CFU g^?1 soil. Conclusion: BOX‐based SCAR markers were useful to find primers for strain‐specific real‐time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation‐independent monitoring methods were lacking. The real‐time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP‐154 and CFN‐535.     

Mycorrhizas in agroforestry: spread and sharing of arbuscular mycorrhizal fungi between trees and crops: complementary use of molecular and microscopic approaches

The spread of arbuscular mycorrhizal (AM) fungi from tree to crop roots was examined by molecular and microscopic methods in a glasshouse study. Growth of Calliandra calothyrsus Meissner trees inoculated with isolates of the AM fungi Glomus etunicatum Becker and Gerdemann and Gigaspora albida Schenck and Smith was monitored over an 18-month period. Three successive ‘intercrops’ of beans or maize were sown at 25, 50 and 75 cm distances from the tree and harvested during this period. At each crop harvest, the distribution of tree and crop roots and the spread of the inoculant fungi were determined using traditional microscopic methods and fungal specific primers. Both inoculants greatly improved the growth of the trees and colonization spread to the crops once the trees were 6 months old. However, benefits of inoculation to crop growth were not observed due to increased competition from the larger inoculated trees growing in a restricted soil volume. Of the two inoculant fungi, Glomus etunicatum appeared to be more mobile as it spread more rapidly, formed higher levels of colonization at increasing distances from the tree and was responsible for most of the mycorrhizal cross-contamination. In contrast, colonization of tree and crop roots by Gigaspora albida was higher nearest the tree. This work demonstrated the benefits of mycorrhizal fungus inoculation for tree growth and confirmed that trees and crops share the same AM fungi. Trees may therefore act as reservoirs of mycorrhizal fungi, either inoculant or indigenous, for surrounding crops or other annual vegetation. It was also shown that tree pruning, the normal practice in agroforestry systems, did not reduce mycorrhizal colonization or prevent spread to crops. However, the slow rates of inoculant spread found here suggest that it may take years before inoculants benefit the growth of crops sown several metres from the tree. The work also demonstrated that microscopic quantification of mycorrhizal colonization and the use of molecular probes to identify specific fungi within roots can complement each other effectively. Molecular probes were more sensitive at detecting mycorrhizal fungi than microscopic methods, but did not discriminate between full mycorrhizal structures and traces of hyphae.     

AMF components from a microbial inoculum fail to colonize roots and lack soil persistence in an arable maize field

Arbuscular mycorrhizal fungi (AMF) are root obligate biotrophs that provide the host with nutrients and pathogen protection, in exchange of photosynthetic products. A decline in AMF diversity can reduce the overall benefit for host plants. A sustainable strategy to re-establish AMF diversity is to supply the target soil with AMF inoculants. After inoculation, it is essential to verify whether the inoculants successfully colonize the host plant and persist, and if the resident AMF community is affected. The AMF components of a microbial inoculum (including other saprotrophs) that was applied to maize were identified and traced in field by 454-pyrosequencing of the partial rRNA 18S gene. In addition, mycorrhizal colonization and plant biomass were monitored in inoculated and non-inoculated maize. The inoculated AMF taxa failed to colonize roots and lacked soil persistence. Nevertheless, the inoculation process reduced species dominance and increased diversity in the pre-existing AMF community. No differences were seen between mycorrhizal colonization in treated and control maize. We suggest that the slightly significant increase in treated plant biomass was potentially due to (i) marginally colonizing inoculated AMF that remained unseen and other saprotroph inoculants applied and/or (ii) the effect of inoculation on the pre-existing AMF community in treated maize roots.     

Comparative analysis of amylolytic lactobacilli and Lactobacillus plantarum as potential silage inoculants

Abstract Two starch-degrading Lactobacillus strains, Lactobacillus amylovorus NRRLB4540 and Lactobacillus amylophilus NCIB11546, were assessed for their potential as silage inoculants. Lactobacillus amylovorus was considered suitable as a silage inoculant for crops which are low in water-soluble carbohydrate, but contain starch, which is unavailable to most conventional silage inoculants. This was on the basis of it exhibiting similar growth characteristics to an inoculant strain of Lactobacillus plantarum and secreting an amylase which was optimally active in the silage pH range. Lactobacillus amylophilus was found to be intolerant of low acid conditions but displayed considerable potential for the industrial production of lactic acid from inexpensive starch-based substrates as it fermented sugars to the desirable L(+) isomer of lactic acid only.     

Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis

A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and WEISSELLA: Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality.     

Immunolocalization of Dinitrogenase Reductase Produced by Klebsiella pneumoniae in Association with Zea mays L.

The endophytic lifestyle of Klebsiella pneumoniae is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by K. pneumoniae strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for K. pneumoniae and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions.     

rep-PCR fingerprinting and taxonomy based on the sequencing of the 16S rRNA gene of 54 elite commercial rhizobial strains

In tropical soils, diversity and biotechnological potential of symbiotic diazotrophic bacteria are high. However, the phylogenetic relationships of prominent strains are still poorly understood. In addition, in countries such as Brazil, despite the broad use of rhizobial inoculants, molecular methods are rarely used in the analysis of strains or determination of inoculant performance. In this study, both rep-PCR (BOX) fingerprintings and the DNA sequences of the 16S rRNA gene were obtained for 54 rhizobial strains officially authorized for the production of commercial inoculants in Brazil. BOX-PCR has proven to be a reliable fingerprinting tool, reinforcing the suggestion of its applicability to track rhizobial strains in culture collections and for quality control of commercial inoculants. On the other hand, the method is not adequate for grouping or defining species or even genera. Nine strains differed in more than 1.03% (15) nucleotides of the 16S rRNA gene in relation to the closest type strain, strongly indicative of new species. Those strains were distributed across the genera Burkholderia, Rhizobium, and Bradyrhizobium.