Dissertations / Theses on the topic 'Microbial inoculants'

A luminescence based marker system was developed for detection of genetically modified Pseudomonas fluorescens and E. coli. During batch growth in liquid culture, luminescence measured by luminometry was directly proportional to biomass concentration and enabled detection of 104 - 106 cells ml-1 of P. fluorescens and 101 cells ml-1 of E. coli, in actively growing cultures. Following inoculation into soil, detection levels were reduced ten-fold. After the subsequent utilisation of available nutrients, activity and luminescence decreased and luminometry then provided a direct, non-extractive means of measuring population activity of lux-marked inocula. Potential luminescence, measured as luminescence following amendment with nutrients, enabled assessment of the rate of reactivation of the lux-marked inocula and quantification of the size of the activatable population. Both these techniques, and traditional techniques, were used to investigate the survival of P. fluorescens and E. coli in soil microcosms. The effect of matric potential and indigenous organisms on luminescence and on survival of P. fluorescens was assessed. Matric potential significantly decreased the activity of both introduced and indigenous populations, but the indigenous population also significantly decreased the activity and biomass concentration of the introduced P. fluorescens population. Use of luminometry as a non-extractive measure of biomass concentration provided qualitative correlation with viable cell concentration, suggesting its potential for rapid enumeration of marked inocula. Reactivation of cells at increased matric stress was decreased, but use of high substrate:cell ratios at -30 kPa produced higher levels of luminescence and may, therefore, improve the use of luminometry as an estimate of biomass.

The concept behind this project was to continue the development of strains of Agrobacterium tumefaciens that were capable of producing pesticidal proteins in response to plant wounding, thereby killing the invading organism. To this end, vir induction was studied in A. tumefaciens and a protocol to elicit the maximum response was developed. In order for this concept to work, it was necessary to determine whether vir induction was occurring at plant wound sites and a method for showing this was developed, the results suggesting that indeed vir induction did occur. The stability of two types of plasmid was also analysed in this bacterium to ascertain how stable the proposed 'microbial inoculant' would be in the field. The results suggested that IncW plasmids should not be used in the final product. The activities of two chitinases from Serratia marcescens were analysed and it was found that both chitinases were effective in controlling some types of fungus. In addition it was found that the expression of chiB in Escherichia coli led to the appearance of a filamentous phenotype at intermediate temperatures. A construct was made that linked the virE promoter to the chiB gene. This plasmid was introduced into A. tumefaciens but did not function as expected. However, other constructs were demonstrated to be inducible although they were only partially successful in controlling the fungi that the strains were assayed against. The Bacillus thuringiensis 5-endotoxin gene, crylA(c), was cloned and various constructs were made to examine the effects of various regions of the native promoter. One constract was made that linked the virB promoter to crylA(c) and this was introduced into A. tumefaciens. The resulting strain was capable of inducible δ-endotoxin expression and was also capable of controlling the larvae of the tobacco homworm, Manduca sexta.

Master of Science
Department of Horticulture, Forestry, and Recreation Resources
Kimberly A. Williams
Liquid organic fertilizers and microbial inoculants of beneficial microorganisms are garnering interest from commercial greenhouse growers who seek to produce crops more sustainably, but research about their efficacy is limited and results are conflicting. This research focused on comparing the effect of microbial inoculant addition in two soilless crop production systems under organic versus conventional fertilization. Two experiments were conducted with impatiens (Impatiens walleriana) in a peat-based substrate and four experiments were conducted with butterhead lettuce (Latuca sativa) in nutrient film technique (NFT) hydroponics. In the impatiens studies, nitrogen, phosphorus, and potassium were incorporated pre-plant equally across treatments using OsmocoteTM, or organic fertilizers Bloodmeal or Feathermeal. An inorganic constant liquid feed (CLF) was also evaluated. Microbial inoculants that contained a variety of beneficial species, including Bacillus spp. and Trichoderma spp. were drench-applied at the beginning of the cropping cycle. Impatiens growth was comparable between the nutrient regimens in one of the studies. CO2 respiration was measured on substrate samples. At a 5X application rate, inoculants contributed to subtle increases in plant growth in organic treatments, but microbial activity was unaffected as measured by CO2 respiration. However, organic nutrient sources contributed to higher CO2 respiration at day 7 of the production cycle compared to inorganic nutrient sources. The hydroponic trials consisted of inorganic and organic nutrient regimens, evaluated with and without microbial inoculant addition. Nutrient analyses and CO2 respiration of the nutrient solutions were collected. Use of inoculants resulted in increased plant growth when used in organic nutrient regimens in some trials. Plant dry weight and CO2 respiration in the inorganic nutrient regimens were increased in certain instances with inoculant addition. No differences in mycorrhizal root colonization were observed in either nutrient regimen with mycorrhizal inoculant addition. Petiole NO3-N concentration of lettuce plants grown with inorganic nutrient sources was greater than that of plants in organic regimens. Organic fertilizers and inoculant products resulted in comparable or positive impacts on plant growth and food crop quality in some treatment scenarios in these studies. The specific circumstances of crop production systems dictate whether plant growth response may occur from inoculant incorporation.

162 leaves printed on single pages, preliminary pages i- vii and numbered pages 1-152. Includes bibliography and abbreviations.
Digitized at 600 dpi grayscale to pdf format (OCR), using a Bizhub 250 Konica Minolta Scanner.
Thesis (PhD(Agric))--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Maize, forage sorghum, lucerne, oats, barley and triticale are the most common silage crops in South Africa, while tropical grasses like Eragrostis curvula and Digitaria eriantha are ensiled to a lesser extent. The aim of this study was to determine the effect of adding a lactic acid bacterial inoculant to E. curvula, D. eriantha, lucerne, forage sorghum, maize and oat silage. The effect of the addition of a lactic acid bacterial inoculant when ensiling E. curvula on the fermentation dynamics during ensiling and the aerobic stability of the silage was determined. The addition of the lactic acid bacterial inoculant to E. curvula at ensiling resulted in a more rapid lowering in pH and improved preservation. Inoculated silage had a higher lactic acid content, less protein breakdown and a lower butyric acid content compared to that of the control silage. Both silages were stable when exposed to air for five days. Digitaria eriantha was ensiled, with or without the addition of a lactic acid bacterial inoculant containing Lactobacillus plantarum. Streptococcus faecium and Pediococcus acidilactici together with the enzymes, cellulase, hemicellulase and amylase. The addition of the inoculant resulted in a more rapid drop in pH, a higher level of lactic acid, an increase in the number of lactic acid bacteria, less protein breakdown and lower numbers of enterobacteria, clostridial spores, yeast and mould compared to the control silage. Digitaria eriantha hay, control and inoculated silage diets were fed to 24 Merino rams (n = 8 per treatment) to determine intake and digestibility. The intake of diets consisting of 90.9% D. eriantha hay, control silage or inoculated silage, differed significantly (p<0.05) at 1395, 1540 and 1848 g DM/day, respectively. The in vivo organic matter digestibility (glkg) of D. eriantha hay, untreated silage and inoculated silage diets was 561, 546, 574, respectively. The addition of the bacterial inoculant when ensiling D.eriantha resulted in better preservation, improved aerobic stability, as well as a higher in vivo organic matter digestibility and intake of D. eriantha silage. The addition of an inoculant or molasses to lucerne (Medicago sativa), ensiled in laboratory silos was investigated. The addition of the additives resulted in an increased preservation rate as indicated by a more rapid lowering of pH, a faster rate of lactic acid production and less protein breakdown compared to control silage. The inoculant was more effective than the molasses in improving the rate of preservation. The aerobic stability of lucerne silage was not affected by inoculation or the addition of molasses. The addition of an inoculant to wilted big bale lucerne silage was studied. The inoculant improved silage quality as indicated by a lower pH, higher lactic acid content, lower ammonia nitrogen content and lower level of butyric acid in inoculated silage compared to the control lucerne silage. The composition of big round bale lucerne silage differed markedly from that of lucerne ensiled in laboratory silos as the former had a higher pH, ammonia nitrogen, butyric acid and acetic acid content and a lower lactic content. Whole crop forage sorghum cultivar FS2 was harvested at the late bloom (20.7% DM) and soft dough (28.9% DM) stages of maturity and ensiled in laboratory silos with the addition of commercial silage inoculants. At both stages of maturity the inoculants caused a more rapid rate of pH decline and a higher amount of lactic acid production. All the silages were well preserved. Silages of the sorghum ensiled at the late bloom stage with all treatments were stable after 5 days of aerobic exposure, whereas sorghum ensiled at the soft dough stage with the addition of the inoculants deteriorated upon aerobic exposure. It is concluded that addition of lactic acid bacterial inoculants to mature sorghum at ensiling might impair the aerobic stability of the silage. The yield, nutritional value and production potential of silage made from twenty one maize hybrids was compared. It was concluded that maize hybrids did differ in metabolizable energy content, rate of digestion, predicted intake and predicted milk production potential. The content of NDF and ADF did not differ between the maize hybrids used in this study and could therefore not be used to predict nutritional value or production potential. Maize was harvested at the hard dough stage and ensiled with or without the addition of a lactic acid bacterial inoculant in laboratory silos and in 210 litre drums. The adding of the inoculant to maize at ensiling did not result in a more rapid drop in pH and higher levels of lactic acid. The intake and growth of South African Mutton Merino lambs fed inoculated and untreated maize silage diets was determined. The average daily gain of lambs fed a diet consisting of either 60% control or inoculated maize silage over a growth period of 60 days was 239 ± 26 and 255 ± 44 g/day, respectively. Although the laboratory study showed very little effect of adding a lactic acid bacterial inoculant to maize at ensiling, lambs tended to consume more of the inoculated silage. In the second study the effect of the addition of a lactic acid bacterial inoculant with an enzyme to maize at ensiling on the fermentation dynamics during ensiling, aerobic stability of the silage, the intake, milk production and milk composition of Jersey cows fed maize silage diets was determined. The inoculant did not result in a more rapid lowering of the pH or a more rapid lactic acid production compared to untreated maize silage made in laboratory silos. Both the control and inoculated maize silages were well preserved. The addition of the inoculant to maize at ensiling improved the palatability, intake and the aerobic stability of maize silage compared to the untreated control maize silage. Milk production, milk composition, live weight and condition score of Jersey cows was not significantly affected by the addition of the inoculant to maize silage. The effect of the addition of an enzyme containing lactic acid bacterial inoculant to big bale oat (Avena sativa, cv Cederberg) silage on silage composition, silage intake, milk production and milk composition of Jersey cows was determined. The crop was cut at the bloom stage, wilted and ensiled in big round bales. The inoculant, Sil-All, was applied during the baling process on half of the bales. Silages were fed to Jersey cows in an intake and milk production study. Both the control and inoculated oat silages were well preserved. The inoculated oat silage had a lower level of butyric acid than the control oat silage. Cows fed the inoculated oat silage produced more (P=O.05) milk (17.7 kg/day) than cows fed the control oat silage (16.7 kg/day). The addition of a lactic acid bacterial inoculant to big bale oat silage improved silage composition and animal performance. This study clearly showed that the composition of silages made in bunker silos under commercial farm conditions differ largely from that of silages made in small scale laboratory silos. When the effect of silage additives on aerobic stability of silage is determined the evaluation should include studies on large scale bunker silages. Evaluation of silage additives should include intake and animal production studies.
AFRIKAANSE OPSOMMING: Mielies, voersorghum, lusem, hawer, gars en korog word algemeen as kuilvoer gewasse benut terwyl tropiese grasse soos Eragrostis curvula en Digitaria eriantha tot 'n mindere mate ingekuil word. Die doel van hierdie studie was om die invloed van 'n melksuurbakterieseinokulant op E. curvula-, D. eriantha-, lusem-, voersorghum-, mielie- en hawerkuilvoer te bepaal. Die invloed van 'n melksuurbakteriese-inokulant op die fermentasiedinamika en die aerobiese stabiliteit van E. curvula-kuilvoer is bepaal. Die toediening van die melksuurbakterieseinokulant tot E. curvula tydens inkuiling het 'n vinniger tempo van pH daling en beter preservering tot gevolg gehad in vergelyking met kontrole kuilvoer. Inokulant behandelde kuilvoer het 'n hoer melksuurinhoud, minder protei'en afbraak en 'n laer bottersuurinhoud as kontrole kuilvoer gehad. Beide kuilvoere was stabiel tydens blootstelling aan lug vir vyf dae. Digitaria eriantha is ingekuil met ofsonder die toediening van 'n melksuurbakteriese-inokulant wat Lactobacillus plantarum. Streptococcus faecium en Pediococcus acidilactici sowel as die ensieme, sellulase, hemisellulase and amilase bevat het. Die inokulant het 'n vinniger tempo van pH-daling, hoer vlakke van melksuur en melksuurbakterie, minder protei'en afbraak en laer getalle van enterobakterie, klostridiale spore, giste and swamme in vergelyking met die kontrole tot gevolg gehad. Digitaria eriantha hooi, kontrole kuilvoer en gei'nokuleerde kuilvoer diete is aan 24 Merino ramme (n = 8 per behandeling) gevoer vir bepaling van inname en verteerbaarheid. Die inname van diete wat uit 90.9% D. eriantha hooi, kontrole kuilvoer of gei'nokuleerde kuilvoer bestaan het, het betekenisvol (p<0.05) verskil en was 1395, 1540 en 1848 gDM/dag, respektiewelik. Die in vivo organiesemateriaal verteerbaarheid (gIkg) vanD. eriantha hooi, kontrole kuilvoer en geYnokuleerde kuilvoer was 561, 546, 574, respektiewelik. Die toediening van die bakteriese-inokulant tydens inkuiling vanD. eriantha het beter preservering, verbeterde aerobiese stabiliteit asook 'n hoer in vivo organiesemateriaal verteerbaarheid van D. eriantha kuilvoer tot gevolg gehad. Die effek van toediening van 'n melksuurbakteriese-inokulant en van molasse tot lusem (Medicago sativa) ingekuil in laboratoriumsilos is ondersoek. Die inokulant toediening en molasse toediening het die tempo van preservering versnel, die pH het vinniger gedaal, melksuur is teen 'n hoer tempo geproduseer en minder proteYen afbraak het plaasgevind in vergelyking met die kontrole kuilvoer. Die tempo van preservering is meer effektief deur toediening van die inokulant verhoog as deur die toediening van molasse. Die aerobiese stabiliteit van lusernkuilvoer is nie beYnvloed deur die toediening van inokulant ofmolasse nie. Die effek van die toediening van 'n melksuurbakteriese-inokulant tot groot rondebaal lusernkuilvoer is ondersoek. Die inokulant het die kwaliteit van die kuilvoer verbeter en het 'n laer pH, hoer melksuur, laer ammoniak stikstofen laer bottersuurinhoud in rondebaallusernkuilvoer tot gevolg gehad in vergelyking met kontrole kuilvoer. Groot rondebaallusernkuilvoer het grootliks verskil van lusernkuilvoer wat in laboratoriumsilos ingekuil is. Die rondebaal kuilvoer het 'n hoer pH, hoer ammoniak-stikstof-, bottersuur- en asynsuurinhoud en 'n laer melksuurinhoud gehad as laboratorium lusernkuilvoer. Voersorghum kultivar FS2 is op die laat blom (20.7% DM) en op die sagte deeg (28.9% DM) stadium met die byvoeging van melksuurbakteriese-inokulante ingekuil in laboratoriumsilos. Toediening van beide inokulante tot sorghum hetop beide die inkuilstadiums gelei tot 'n vinniger tempo van pHdaling en meer melksuurproduksie. Aile kuilvoere insluitend die kontrole kuilvoer was goed gepreserveer. Kontrole sowel geYnokuleerde sorghumkuilvoer ingekuil op die laat blomstadium was stabiel tydens aerobiese blootstelling vir 5 dae. Sorghumkuilvoer ingekuil op die sagtedeegstadium met die byvoeging van inokulante was onstabiel tydens aerobiese blootstelling. Die toediening van melksuurbakteriese-inokulante tot sorghum wat op die sagtedeegstadium ingekuil word kan aerobiese stabiliteit van die kuilvoer grootliks benadeel. Die opbrengs, voedingswaarde en produksiepotensiaal van kuilvoer gemaak van 21 mielie hibriede is vergelyk. Verskille in metaboliseerbare energie inhoud, tempo van vertering, voorspelde inname en voorspelde melkproduksie het tussen mielie hibriede voorgekom. Die neutraalbestandevesel- en suurbestandeveselinhoud het nie verskil tussen hibriede nie en derhalwe kon dit nie gebruik word om voedingswaarde ofproduksiepotensiaal te beraam rue. Mielies is op die hardedeegstadium met of sonder die toediening van 'n melksuurbakterieseinokulant in laboratoriumsilos en 210 liter dromme ingekui!. Die toediening van die inokulant het geen invloed op tempo van pH-daling ofproduksie van melksuur gehad nie. Die inname en groei van SA Vleismerino lammers wat 'n dieet bestaande uit 60% kontrole of inokulant behandelde mieliekuilvoer ontvang het, is bepaa!. Die gemiddelde daaglikse toename van lammers was 239 ± 26 and 255 ± 44 g/dag vir die kontrole en inokulant mieliekuilvoer dieet respektiewelik. Alhoewel die laboratoriumstudie weinig verskille tussen die kontrole en die geYnokuleerde mieliekuilvoer getoon het, het lammers geneig om meer van die geYnokuleerde mieliekuilvoer in te neem. In die tweede studie met mieliekuilvoer is die effek van toediening van 'n melksuurbakteriese-inokulant met ensieme, op die ferrnentasiedinamika tydens inkuiling, die aerobiese stabiliteit van mieliekuilvoer asook die inname, melkproduksie en melksamestelling van Jersey koeie bepaa!. Die inokulant het nie die tempo van pH daling en produksie van melksuurverhoog nie en beide kontrole en geYnokuleerde mieliekuilvoerwas goed gepreserveer. Die toediening van die inokulant tot mieliekuilvoer het die smaaklikheid, inname en die aerobiese stabiliteit van mieliekuilvoer verhoog in vergeiyking met kontrole mieliekuilvoer. Melkproduksie, melksamestelling, liggaamsmassa en kondisiepunt van Jersey koeie is nie betekenisvol beYnvloed deur die toediening van die inokulant tot mieliekuilvoer nie. Die effek van die toediening van 'n ensiem bevattende melksuurbakteriese-inokulant tot groot rondebaal hawer (Avena sativa, cv Cederberg) kuilvoer op die samestelling van kuilvoer, kuilvoerinname, melkproduksie en melksamestelling van Jersey koeie is bepaa!. Die gewas is gesny op die blomstadium, verwelk en as rondebaalkuilvoer gepreserveer. Die inokulant, Sil-All, is tydens die baalproses toegedien op die helfte van die bale. Kuilvoere is aan Jersey koeie gevoer in 'n inname en melkproduksiestudie. Beide die kontrole en geYnokuleerde hawerkuilvoer was goed gepreserveer. Die bottersuurinhoud van geYnokuleerde hawerkuilvoer was laer as die van die kontrole hawerkuilvoer. Koeie wat geYnokuleerde hawerkuilvoer gevoer is het meer (P=0.05) melk (17.7 kg/dag) geproduseer as koeie wat kontrole hawerkuilvoer ontvang het (16.7 kg/dag). Die toediening van 'n melksuurbakteriese-inokulant het kuilvoer kwaliteit en diereproduksie verbeter. Hierdie studie wys duidelike verskille uit tussen kuilvoer wat in bunkersilos onder kommersiele toestande ingekuil is, en kuilvoer wat in laboratoriumsilos gemaak is. Wanneer die effek van kuilvoerbymiddels op die aerobiese stabiliteit van kuilvoer bepaal word behoort finale evaluasie gedoen te word op kuilvoer gemaak in bunkersilos soos onder plaastoestande plaasvind. Evaluasie van kuilvoerbymiddels behoort inname en diereproduksiestudies in te sluit.

Thesis (M. Tech.) -- Central University of Technology, Free State, 2010
This study evaluated the effects of two bacterial inoculants on the fermentation quality and aerobic stability of ensiled potato hash (PH) and on digestibility, growth performance and carcass characteristics of growing pigs fed the ensiled PH. In the first experiment, 700 grams of PH, was mixed with 300g wheat bran (as is basis) and ensiled in 1.5 litre anaerobic jars. The mixture had a dry matter (DM) of 41.3 g/kg, 78.05 g/kg DM of water-soluble carbohydrates (WSC) and a pH of 6.87 at day 0 and was treated with Bonsilage forte, BF, a heterofermentative LAB, Lalsil Fresh lactobacillus bachneri, LFLB, a homofermentative LAB or without LAB inoculant (control). Triplicate samples per treatment were collected on days 0, 3, 10, 21 and 45 post-ensiling and analysed for DM, WSC, pH, lactic acid (LA), acetic acid (AA), butyric acid (BA), propionic acid (PA) and ammonia-N. On day 45, the silage was subjected to aerobic exposure for 5 days and CO2 production was measured. In the second and third experiments, potato hash-wheat bran mixtures of 7:3 ratio were produced and ensiled in 210 litre drums that were kept at a 250C environment. The second experiment involved an apparent nutrients digestibility study using 30 female (Large White x Landrace crossbred) pigs (30 2 kg live weight), which were selected and housed individually. A cross-over design was applied with two periods and 30 animals fed 10 diets. Three pigs were given one diet per period. The diets were: commercial feed (control); 20, 40, and 60% dietary inclusion of potato hash silage (untreated potato hash silage, UPHS; Lalsil Fresh treated potato hash silage, LFLBPHS, and Bonsilage forte treated potato hash silage, BFPHS). Each experimental period lasted for eleven days with seven days being for adaptation to each diet and four days for collection of faeces before crossing over to the next treatment. Pigs were fed twice per day at 8:00 and 15:00, with the daily allowance equally divided between the two meals. Faecal samples were weighed and 10% of the total collection was taken and kept in a freezer at -180C. Prior to chemical analysis, individual samples of faeces were thawed and pooled for pigs within periods. Water was made available at all times through drinking nipples. The third experiment was a pig growth study using sixty-four crossbred pigs (Large White x Landrace) consisting of 32 males and 32 females (30.4±2.3kg live weight). The pigs were randomly allocated to four diets in a 4 x 2 (treatment x sex) factorial experiment in a completely randomised design. The diets contained up to 40% potato hash silage (PHS) and were: control (commercial diet - no silage), UPHS, BFPHS and LFLBPHS. Each experimental unit consisted of two pigs and each treatment was replicated 8 times. Pigs were fed on one of the four diets until they reached a slaughtering weight of 60 kg. Warm carcass weights were determined immediately after slaughter. After an overnight chill storage at 4.30C, cold carcass weights were determined. Carcass length was taken on hanging carcasses by measuring from the pelvic bone to the first thoracic vertebra, using a measuring tape. Backfat thickness was also taken on cold carcass at P2 (45 mm from midsection) between the 3rd and 4th rib on the left side of the pigs. Drip loss was also calculated for hanging carcasses by determining the weight loss after the overnight chill. Results from experiment 1 showed that both inoculants (LFLB and BF) reduced (P<0.05) the silage pH while increasing the LA content of silage compared to the control. The concentrations of propionic acid, butyric acid and ammonia-N were not affected by inoculation. When exposed to air, BF and LFLB reduced (P<0.05) CO2 production compared to the control. Results of the second experiment showed that daily intakes of organic matter (OM), crude protein (CP), ether extract (EE), acid detergent fibre (ADF), neutral detergent fibre(NDF), and gross energy (GE) were different (P<0.05) between diets. There were no differences (P<0.05) in digestibility of DM, EE, and DE among the treatments. However, diets containing 60% PHS had lower (P<0.05) NDF and ADF digestibility compared to diets containing less PHS. Furthermore, pigs on the control diet had higher (P<0.05) final body weight, average daily gain (ADG) and better feed conversion ratio (FCR) compared to those fed on other diets. Results of the third experiment showed that, pigs that were fed the control diet had higher (P<0.05) slaughter weight than pigs that were fed on diets containing PHS. There were no differences (P>0.05) on warm and carcass weight between diets containing PHS. Control had higher (P<0.05) dressing percentage than the other treatments. Drip loss percentage, backfat thickness, gastrointestinal tract (GIT), lungs and heart did not differ (P<0.05) between treatments. The effects of gender was not significant within treatments on slaughter weight, warm and cold carcass weights, dressing percentage, carcass length, backfat thickness, drip loss, GIT, lungs and heart. There were no gender by diet interactions (P>0.05) on warm carcass weights, cold carcass weights, drip loss percentage, carcass length, backfat, intergastral, lungs and heart. However, gender by diet interaction (P<0.05) occured on slaughter weight and dressing percentage. It was concluded that, inoculation improved both the fermentation and aerobic stability of PHS. Furthermore, there was no advantage in using LAB inoculants in ensiling potato hash on the growth performance or meat characteristics of growing pigs. However, further work is needed to evaluate the effects of higher dietary inclusion levels (> 60 %) of PHS on pig growth and reproductive performance.

With shifting preferences of consumers towards healthier food, organic food demand has been on the rise for the past two decades. This increased demand has created an opportunity for farmers to shift from conventional to organic production. However, there are risks and uncertainties associated with organic farming. The management of an organic farm in the absence of organic-based disease and pest suppressing strategies constrains farmers from adopting organic vegetable production. The use of cover crops to control soil-borne diseases and suppress weeds and other pests has increased because of its sustainable and environmental friendly nature. This study of the economic impact of the cover crops on organic tomato production in the three states Ohio, New York, and Maryland showed mixed results. In Maryland, mixed forage radish and hairy vetch was projected to have a net present value over 15 years that was $1.53 million higher than single species hairy vetch, assuming maximum adoption level of 50 percent. In New York, mixed rye and turnip gave the higher return with a net present value of $2.61 million. In Ohio, the highest projected return was from mixed hay compared to hairy vetch with a net present value of $3.12 million when used without adding compost amendments. In Maryland and New York when bare ground was also used as a control, only mixed forage radish and hairy vetch in Maryland produced better returns compared to bare ground. A probit regression assessing the factors affecting the decision to adopt mixed species green manure technology indicated that farmer experiences in organic production, farmer age, access to the internet access, and farmers’ perceptions about the benefits of using mixed species green manures were significant factors. Each variables and factors except age had a positive influence. Similarly, probit results for microbial inoculants indicated that education, gender, and access to the internet were significant determinants, and had a negative effect on the probability of adoption. Access to the internet was significant for both mixed species green manures and microbial inoculants but with opposite sign, positive for mixed species green manures and negative for microbial inoculants.
Master of Science

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Some Brazilian states, especially the Northeast, has its economy based on the cultivation of sugarcane, which depends on the success of this activity for the subsequent performance of its economy. In this context, priority is performing prospecting work diazotrophs and research to evaluate the potential of biological nitrogen fixation (BNF) in sugarcane under regional condition, applying technologies that enable the sustainable production of this crop. With that aimed to isolate and make characterizations morphophysiological, biochemistry and molecular endophytic bacteria stems and leaves of three varieties of sugarcane (RB1011, RB92579 and RB867515) and to evaluate the response of the cane sugar and the potential of BNF, the efficiency of inoculation of these bacteria as the productivity of stalks and technological qualities of sugarcane in the cycles of plant cane, cane ratoon and cane ressoca as support for sustainable crop production. 56 isolates were endophytic, being 62.5% and 37.5% of the stem of the leaf. We used molecular BOX-PCR technique that allows to differentiate into isolated sections 34 bands, for the formation of five distinct clusters that were selected for biochemical and / or physiological tests. These isolates, 66% were able to solubilize inorganic phosphate; 26% to produce urease, proteases produce 34%; 17% amylase to produce; 68% to produce lipase; 14% to produce chitinase; 17% of producing siderophore; 71% of produce ACC deaminase; 54% to produce N-acyl homoserine lactone (AHL); 51% to produce catalase and 23% showed hydrolytic ability to degrade gelatin; 97% reduced acetylene (ARA) with values between 4.14 and 7.84 nmol C2H4 / hour. To sequence the 16S rRNA ribosomal the presence of seven different genres, 46% identity was observed with the genus Bacillus, 20% to Paenibacillus, 14% Burkholderia, 6% Herbaspirillum, Pseudomonas and Methylobacterium and 3% Brevibacillus . Biochemical and molecular characterization of endophytic isolates stem and leaf of sugarcane revealed a diversity of bacteria colonizing this plant, with biotechnological potential to be used as biofertilizers. Three isolated by the nitrogenase activity and together with the responses of biochemical tests were selected to compose the microbial inoculant that was tested in the field for three years comprising the cycles of sugar cane plant, soca and ressoca the variety of sugarcane RB 92579 . We used the experimental randomized block design in a factorial 10x3, consisting of ten treatments (IPACC9 (Herbaspirillum seropedicae), IPA-CC33 (Pseudomonas sp.), IPA-CF65 (Bacillus Megaterium), IPA-CC9 + IPA-CC33, IPA-CC9 + IPA-CF65, IPA-CC33 + IPA-CF65, IPACC9 + IPA-CC33 + IPA-CF65, mix Embrapa (BR1140 (Azospirillum amazonense) BR11175 (H. seropedicae) BR11192 ( H. rubrisubalbicans) BR11284 (Gluconacetobacter diazotrophicus) and BR11364 (Burkholderia tropica), nitrogen witness (TN) and absolute control (TA)), three crop cycles (plant cane, cane and ratoon cane ressoca) with four replications totaling 40 plots for cultivation were evaluated the following parameters:.plant height, stem diameter, shoot dry weight, total nitrogen leaves +3, fresh stalk yield, sugar yield per hectare (TPH) content solids (° Brix) soluble fiber, total recoverable sugar (ATR) and estimation of the contribution of BNF nutrition of sugarcane by the technique of natural abundance of 15N isotope. Inoculation promoted significant gains compared to TA for the inoculated treatments was up to 57.31%. It found increases in productivity fresh stalk of up to 14 Mg ha-1 compared to TN, but not statistically differing from the other treatments. The similar behavior of the treatments inoculated with TN who received 60 kg ha-1 N, infers in low economic and environmental cost, and gains importance by the need to develop more sustainable agricultural practices to improve the quality of life of the population.
Alguns estados brasileiros, especialmente da região Nordeste, têm sua economia baseada na cultura da cana-de-açúcar, que depende do êxito dessa atividade para o consequente desempenho de sua economia. Nesse contexto, é prioritário realizar trabalhos de prospecção de bactérias diazotróficas e de pesquisas que avaliem a potencialidade da fixação biológica do nitrogênio (FBN) na cana-de-açúcar, sob condição regional, aplicando tecnologias que permitam a produção sustentável desta cultura. Com isso, objetivou-se isolar e efetuar caracterizações morfofisiológica, bioquímica e molecular de bactérias diazotróficas endofíticas de colmos e folhas de três cultivares de cana-de-açúcar (RB1011, RB92579 e RB867515), assim como avaliar a resposta da cana-de-açúcar quanto ao potencial de FBN, a eficiência da inoculação destas bactérias quanto à produtividade de colmos e qualidades tecnológicas de cana-de-açúcar nos ciclos da cana-planta, cana-soca e cana-ressoca como suporte a uma produção sustentável da cultura. Foram obtidos 56 isolados endofíticos, sendo 62,5% do colmo e 37,5% da folha. Utilizou-se a técnica molecular de BOX-PCR que permitiu diferenciar os isolados em 34 perfis de bandas, pela formação de cinco agrupamentos distintos, que foram selecionadas para os testes bioquímicos e/ou fisiológicos. Desses isolados, 66 % apresentaram capacidade de solubilizar fosfato inorgânico; 26 % de produzirem urease, 34 % de produzirem proteases; 17 % de produzirem amilase; 68 % de produzirem lipase; 14 % de produzirem quitinase; 17 % de produzirem sideróforo; 71 % de produzirem ACC deaminase; 54 % de produzirem N-acil-homoserina lactona (AHL); 51 % de produzirem catalase e 23 % apresentaram capacidade hidrolitica de degradarem a gelatina; 97 % reduziram acetileno (ARA) com valores entre 4,14 e 7,84 nmol C2H4/hora. Para o sequenciamento do gene 16S rRNA ribosomal, foi verificado a presença de sete diferentes gêneros, 46% de identidade com o gênero Bacillus , 20% com Paenibacillus, 14% com Burkholderia, 6 % com Herbaspirillum, Pseudomonas e Methylobacterium e 3% com Brevibacillus. A caracterização bioquímica e molecular dos isolados endofíticos de colmo e folha de cana-de-açúcar revelou uma diversidade de bactérias colonizando esta planta, com potencial biotecnológico para serem usadas como biofertilizantes. Foram selecionados três isolados pela atividade da nitrogenase e em conjunto com as respostas dos testes bioquímicos para compor o inoculante microbiano que foi avaliado em campo durante três anos compreendendo os ciclos da cana planta, soca e ressoca na variedade de cana-de-açúcar RB 92579. Utilizou-se o delineamento experimental em blocos casualizados, em esquema fatorial 10x3, constituído de dez tratamentos (IPA-CC9 (Herbaspirillum seropedicae), IPACC33(Pseudomonas sp.), IPA-CF65 (Bacillus Megaterium), IPA-CC9 + IPA-CC33, IPACC9 + IPA-CF65, IPA-CC33 + IPA-CF65, IPA-CC9 + IPA-CC33 + IPA-CF65, Mistura Embrapa (BR1140 (Azospirillum amazonense), BR11175 (H. seropedicae), BR11192 (H. rubrisubalbicans), BR11284 (Gluconacetobacter diazotrophicus) e BR11364 (Burkholderia tropica), testemunha nitrogenada (TN) e testemunha absoluta ( TA)), três ciclos de cultivo (cana-planta, cana-soca e cana-ressoca) com quatro repetições, totalizando 40 parcelas por cultivo. Foram avaliados os seguintes parâmetros: altura de plantas, diâmetro do colmo, massa seca da parte aérea, nitrogênio total das folhas +3, produtividade do colmo fresco, rendimento de açúcar por hectare (TPH), teor de sólidos solúveis totais (°Brix), fibra, açúcar total recuperável (ATR) e estimativa da contribuição da FBN na nutrição da cana-de-açúcar pela técnica de abundância natural do isótopo 15N. A inoculação promoveu ganhos significativos em comparação a TA para o diâmetro do colmo e ATR, para o cultivo canaressoca. A contribuição da FBN nos tratamentos inoculados foi de até 57,31%. Constatou-se incrementos na produtividade do colmo fresco de até 14 Mg.ha-1 em comparação a TN, porém não diferenciando estatisticamente dos outros tratamentos. O comportamento semelhante dos tratamentos inoculados com a TN que recebeu 60 kg.ha‑1 de N, infere em baixo custo econômico e ecológico, e ganha importância pela necessidade para o desenvolvimento de práticas agrícolas mais sustentáveis para melhorar a qualidade de vida da população.

Thesis (MSc Food Sc)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: The start-up period of Upflow Anaerobic Sludge Bed (UASB) reactors can significantly be reduced by enhancing the time-consuming granulation process through the batch cultivation of anaerobic granular sludge and thus seeding the reactor with this cultivated granular sludge, instead of raw anaerobic sludge. The precise mechanism for granule formation is not well known, but it is believed that extracellular polymers (ECP) play a critical role in the granulation process. Information on the precise role of ECP is also limited and no universal standardised method for ECP extraction is used at present. Therefore, comparison of results from different researchers has to be made with great caution. The objectives of this study were to evaluate an ECP extraction methód so as to optimise the extraction time, and then to correlate ECP composition of UASB granules with granule metabolic activity. The impact of changes in the environmental conditions, such as sludge sources (Paarl and Kraaifontein-sludge), carbon growth substrates (yeast extract lactate, glucose medium and fruit cocktail effluent) and batch cultivation techniques (roller-table and shake-waterbath), on batch cultivation studies was also evaluated in terms of granule activity, ECP composition and granule formation. A physical extraction method was used to quantify the ECP content of UASB granules from six different sources. The optimal extraction time was taken as the time needed before cell lysis took place, and before intracellular material started contributing to the ECP content of the granules. It was concluded that the ECP composition was affected by the wastewater composition fed to the original UASB reactors, It was also found that the activity test results could be used to indirectly predict the activity of the different trophic groups present in the UASB granules. A correlation was found between the activity test results and the total ECP content, and this showed that the granules with the higher ECP yields exhibited greater biogas (SB) and methanogenic (SM) activities. However, based on the activity data and total ECP content, it appeared that a protein:carbohydrate ratio < 1 affected the activity of the granules, The sludge source used as inoculum for batch cultivation of anaerobic granular sludge had a significant effect on granule formation. The use of a pre- granulated raw anaerobic sludge, such as the Paarl-sludge, resulted in a greater increase in granule numbers at the end of the cultivation period. The acetic acid activity profiles showed that the acetoclastic methanogens that are involved in initiation of granulation by nucleus formation, were inactive or absent in the different batch systems, with the exception of the roller-table glucose cultivated Kraaifontein-sludge (RKG) batch system. The addition of glucose as carbon growth substrate for batch cultivation not only enhanced the activity of the acidogenic population, but also led to the establishment to a greater variety of granule trophic groups within all the glucose cultivated batch systems. The addition of fruit cocktail effluent as carbon substrate enhanced ECP production in the Paarl-sludge cultivated batch systems. However, the addition of carbon substrates showed no discernible trend on granule formation itself. The roller-table cultivation technique resulted in the higher increase in granule numbers, and it was speculated that the more vigorous shake-waterbath technique probably shortened the contact time between biomass and substrate. Large variations in the ECP composition of the different batch systems were found, and these were ascribed to the composition heterogeneity of different sludges. For future studies, it is advisable to characterise sludge, both chemically and microbiologically before using as inoculum. The selection of an appropriate sludge inoculum should then lead to optimisation of the granulation process.
AFRIKAANSE OPSOMMING: Die aanvangsperiode van "Upflow Anaerobic Sludge Bed" (UASB) bioreaktors kan noemenswaardig verminder word deur die tydsame granulasie proses te versnel deur die vooraf lot-kweking van anaërobe granulêre slyk waarmee 'n reaktor dus geïnokuleer kan word in plaas van rou anaërobe slyk. Die presiese meganisme van granulevorming is nog nie welbekend nie, maar daar word beweer dat ekstrasellulêre polimere (ECP) wel 'n kritiese rol speel in die granulasie proses. Inligting; oor die presiese rol van ECP is ook nog beperk, en tans word daar nog , geen universele standaard metode vir ECP-ekstraksie gebruik nie. Gevolglik moet resultate vanaf verskeie navorsers met groot omsigtigheid vergelyk word. Die doelwitte van hierdie studie was om 'n ECP ekstraksiemetode te evalueer deur die ekstraksietyd te optimiseer, en dan te korreleer met die ECP samestelling en metaboliese aktiwiteit van die UASB granules. Die inwerking van veranderinge in omgewingskondisies, soos slykbronne (Paarl- en Kraaifontein-slyk), koolstofbronne (gisekstrak-Iaktaat-, glukose-medium en vrugtekelkie-uitvloeisel) en lot-kwekingstegnieke (rol-tafel en skud-waterbad) op lot-kweking studies in terme van granule aktiwiteit, ECP-samestelling en granulevorming is ook ondersoek. 'n Fisiese ekstraksie metode is gebruik om die ECP-inhoud van UASB granules vanaf ses verskillende bronne te bepaal. Die optimale ekstraksietyd is geneem as die tyd benodig voordat sellise sal plaasvind en die intrasellulêre materiaal 'n bydrae sal lewer tot die ECP-inhoud van granules. Dit is afgelei dat die ECP-samestelling beïnvloed word deur die samestelling van die afvalwater wat vir die oorspronklike UASB bioreaktors gevoer is. Voorts is gevind dat die aktiwiteitstoets resultate indirek gebruik kan word vir die voorspelling van aktiwiteit van die verskillende trofiese groepe wat in die UASB granules teenwoordig is. 'n Korrelasie is gevind tussen die aktiwiteitstoets resultate en die totale ECP-inhoud wat aangedui het dat granules met hoër ECP opbrengste, beter biogas (SB) en metanogeniese (SM) aktiwiteit getoon het. Volgens die aktiwiteitsdata en totale ECP-inhoud het dit egter geblyk dat 'n proteïen:koolhidraat verhouding < 1 die aktiwiteit van granules beïnvloed het. Die slykbron wat as inokulum gebruik is vir lot-kweking van' anaërobiese granulêre slyk het 'n noemenswaardige invloed gehad op granulevorming. Die gebruik van 'n rou anaërobe slyk wat reeds 'n mate van granulasie getoon het, soos die Paarl-slyk, het 'n hoër toename in granule getalle aan die einde van die kwekingsperiode teweeg gebring. Die asynsuur aktiwiteitsprofiele het aangedui dat die asetoklastiese metanogene, wat hoofsaaklik betrokke is by inisiëring van granulasie deur kernvorming, onaktief of afwesig was in al die verskillende lotsisteme, met die uitsondering van die roltafel glukose-gekultiveerde Kraaifonteinslyk (RKG) lot-sisteem. Die toevoeging van glukose as koolstofbron vir lot-kweking het nie alleenlik die aktiwiteit van die asidogene populasie verhoog nie, maar het ook bygedra tot die vestiging van 'n groter verskeidenheid van granule trofiese groepe. Die toevoeging van vrugtekelkie-uitvloeisel as koolstofbron het die produksie van ECP verhoog in die Paarl-slyk gekweekte lot-sisteme. Die toevoeging van koolstofsubstrate het egter geen merkbare verandering getoon in granulasie opsigself nie. Die rol-tafel kwekingstegniek het 'n hoër toename in granule getalle tot gevolg gehad, en dit is gespekuleer dat die meer kragtige skud-waterbad tegniek waarskynlik die kontaktyd tussen die substraat en biomassa verkort het. Groot variasies is gevind in die ECP-samestelling van die verskillende lotsisteme, en dit is toegeskryf aan die heterogene samestelling van die verskillende slyke. Vir toekomstige navorsing is dit raadsaam om slyk voor gebruik as inokulum beide chemies en mikrobiologies te karakteriseer. Die keuse van 'n geskikte slyk-inokulum sal bydra tot die optimisering van die granulasie proses.

The use of silage additives can improve the reliability of the ensilage process. The fact that chemical additives are both highly corrosive to expensive farm machinery and hazardous to handle has prompted the development of alternative silage additives. Bacterial inoculants were developed to provide a source of homofermentative lactic acid bacteria. The objective of this study was to develop a bacterial silage inoculant that would improve the efficiency of ensilage of a crop. An inoculant termed SilCare II was developed. SilCare II, comprising a ratio of three L.plantarum L71 cells to every one P.pentosaceus P826, was always applied in the presence of molasses at the rate of 9 ml kg^-1 herbage. The freeze-drying of SilCare II in the presence of rehydrated skimmed milk was found to be the most successful protectant for preserving the cultures, both during the freeze-drying process and over a long storage period. Freeze-dried SilCare II preserved herbage more successfully than a commercially available inoculant, Lactomol, or an untreated control. Improvements in the efficiency of the ensilage process were achieved by increasing the application rate of the inoculant to the herbage. However, large scale ensilage experiments indicated that the inoculant had little effect on the fermentation of herbage in bunker silos. Animals trials using grey-faced wether lambs indicated that SilCare II did not significantly improve the nutritional quality of the silage.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Objetivou-se avaliar a estabilidade aeróbia de silagens de planta e de grãos úmidos de milho com a utilização de inoculante microbiano, bem como o uso de filme plástico de baixa permeabilidade ao oxigênio associado ao filme convencional. Para tanto foram conduzidos três experimentos. No primeiro e segundo experimentos os objetivos foram avaliar os efeitos de doses de Lactobacillus buchneri (5x104, 1x105, 5x105 e 1x106 UFC/ g) sobre as características fermentativas, a dinâmica microbiológica, a estabilidade aeróbia e o valor nutricional de silagens de planta e de grãos úmidos de milho. No terceiro experimento, objetivou-se avaliar o efeito do filme plástico de baixa permeabilidade ao oxigênio associado ao filme convencional sobre a perdas na armazenagem e a estabilidade aeróbia de silagem de planta de milho, em silo tipo superfície. O L. buchneri é eficiente no controle de leveduras e fungos filamentosos, assim como melhora a estabilidade aeróbia da silagem de planta e de grãos úmidos de milho e não afeta o valor nutricional. O filme de baixa permeabilidade ao oxigênio associado ao filme convencional é eficiente na manutenção das características fermentativas, no controle da população de leveduras e na redução de perdas, principalmente quando a fatia de retirada é menor
Aimed to evaluate the aerobic stability of plant and of high moisture corn silage with the use of microbial inoculant and oxygen barrier film associated with conventional film. Therefore, three experiments were conducted. In the first and second experiments the objectives were to evaluate the effects of Lactobacillus buchneri doses (5x104, 1x105, 5x105 and 1x106 CFU/g) on the fermentation characteristics, microbial dynamics, aerobic stability and nutritional value of plant and of grain moisture corn silage. The third experiment aimed to evaluate the effect of oxygen barrier film associated with conventional film under the storage losses and aerobic stability of plant corn silage in a horizontal silo. The L. buchneri is effective in control of yeasts and molds, and improve the aerobic stability of plant and of high moisture corn silage and do not affect nutritional value. The oxygen barrier film is effective in maintaining the fermentation characteristics, in yeasts population control and loss reduction, mainly when the feedout rate is lower

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A altura da planta apresenta uma alta correlação com a estrutura do pasto. Por esta razão, levar em consideração a altura da pastagem tem sido uma estratégia muito melhor no gerenciamento rotativo de estocagem em pastagens tropicais do que considerando dias predefinidos de crescimento. Da mesma forma, a altura da pastagem pode ser usada para determinar o momento em que as gramíneas tropicais apresentam os melhores parâmetros de ensilabilidade. Este estudo teve como objetivo identificar a altura de crescimento máximo da planta de Panicum maximum cv. Mombaça (Capim Mombaça) que fornece a maior capacidade de fermentação (CF) e definir a combinação de aditivos que melhora a composição química da silagem. Dois experimentos foram realizados em Selvíria, MS, Brasil, de 2015 a 2016. O primeiro ano foi usado para identificar a CF mais alta e o segundo ano foi usado para identificar a melhor combinação de oito aditivos (polpa cítrica (PC), homo e heterofermentativo LAB, suas combinações e controle). As análises estatísticas foram realizadas com SAS (P <0,05) e um contraste foi definido como silagem com PC versus silagem sem PC. A altura de 130 cm resultou na maior CF (31,01). As silagens inoculadas com PC apresentaram melhor qualidade do que as silagens sem PC, devido à alta proteína bruta (8,3 vs. 7,3% MS), recuperação de MS (98,6 vs. 93,3% MS) e baixo valores de pH (3,92 vs. 4,91) e NH3-N (2,49 vs. 14,73% de N total). A altura da planta é um parâmetro consistente para determinar o tempo de ensilagem do Capim Mombaça e a inclusão do PC é necessária para aumentar a qualidade da silagem.
The sward height presents a high correlation with the pasture sward structure. For this reason, taking the sward height into account has been a much better strategy in rotational stocking management in tropical grasslands than considering predefined days of growth. Similarly, the sward height could be used to determine the moment when the tropical grasses present the best ensilability parameters. This study aimed to identify the sward height of Panicum maximum cv. Mombaça (Guinea grass) that provides the highest fermentability coefficient (FC) and to define the combination of additives that improves the chemical composition of silage. Two trials were carried out in Selvíria, MS, Brazil from 2015 to 2016. The first year was used to identify the highest FC and the second year was used to identify the best combination of eight additives [citrus pulp (CIP), homo and heterofermentative LAB, their combinations, and control]. Statistical analyses were performed using SAS (P < 0.05) and one contrast was defined as silage with CIP versus silage without CIP. The height of 130 cm resulted in the highest FC (31.01). Silages inoculated with CIP had a better quality than silages without CIP, due to the high crude protein (8.3 vs. 7.3 % DM), DM recovery (98.6 vs. 93.3 % DM), and the low pH (3.92 vs. 4.91), and NH3-N values (2.49 vs. 14.73 % total N). The sward height is a consistent parameter to determine the time of ensiling Guinea grass and the inclusion of CIP is necessary to raise the silage quality.
FAPESP: 2014/10356-0

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Objetivou-se neste trabalho avaliar os efeitos de dietas contendo silagem inoculada com Lactobacillus plantarum e Bacillus subtilis e suplementadas ou não com amilase sobre a digestibilidade aparente, fermentação ruminal e síntese de proteína microbiana em carneiros, assim como o desempenho e qualidade de carne de cordeiros. Para tanto, dois estudos foram conduzidos, no quais os animais receberam um dos quatro tratamentos (dietas): 1) silagem de milho não inoculada sem adição de amilase na mistura total da ração (MTR); 2) silagem de milho não inoculada e amilase adicionada na MRT; 3) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis, sem adição de amilase; 4) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis e amilase adicionada na MRT. A enzima utilizada foi a amilase numa taxa de aplicação de 2 g de produto / kg de matéria seca (MS) da dieta (602 unidade dextrinizante (UD) / kg de MS da dieta). A suplementação com amilase em dietas contendo silagem não inoculada aumentou (P=0,045) o consumo de matéria seca dos carneiros quando comparados com aqueles alimentados com silagem não inoculada sem suplementação com amilase (1,311 vs. 1,066 g/d), mas não diferiu dos outros tratamentos. A digestibilidade aparente da MS, MO, PB, FDN e EB aumentou (P<0,01) nos carneiros alimentos com silagem inoculada ou suplementados com amilase, sem interações entre os tratamentos. Os animais alimentados com dietas contendo silagem não inoculada e suplementados com amilase apresentaram alta proporção de ácido propiônico e baixa de ácido acético, e consequentemente baixa relação de aceitoc:propiônico. A síntese de proteína microbiana tendeu a ser maior (P=0,097) nos carneiros alimentados com silagem não inoculada e suplementados com amilase e também nos que receberam dieta contendo silagem inoculada sem suplementação com amilase (8,01; 8,05 g/d, respectivamente). Entretanto, nenhum efeito foi verificado na eficiência de síntese de proteína microbiana. No segundo estudo, cordeiros alimentados com silagem inoculada apresentaram maior consumo de FDN (P=0,019) do que aqueles alimentados com silagem não inoculada (266,5 vs. 245,0 g/d). Cordeiros que receberam dieta contendo silagem inoculada apresentam maior ganho de peso diário (P=0,019) quando comparados àqueles alimentados com silagem de milho não inoculada (232,5 vs. 211,5). A inoculação da silagem aumentou (P<0,05) o conteúdo de ácidos graxos saturados (AGS) e diminuiu (P<0,05) o conteúdo de ácidos graxos insaturados (AGI) (47,55 vs. 46,21% e 52,44 vs. 53,79%, respectivamente), e consequentemente, diminuiu a relação UFA:SFA. A suplementação com amilase no momento da alimentação tendeu (P<0,10) a diminuir a relação AGPI:AGS (0,14 vs. 0,16). O uso de amilases em dietas contendo silagem de milho inoculada não resultou em respostas positivas na digestibilidade e síntese de proteína microbiana de carneiros, bem como não alterou as características de carcaça e qualidade de carne de cordeiros. O uso de dietas contendo silagem de milho inoculada com L. plantarum e B. subtilis e não suplementadas com amilase, aumentou o ganho de peso de cordeiros.
This trial aimed to evaluate the effects of diets containing corn silage inoculated with Lactobacillus plantarum and Bacillus subtilis and supplemented or not with amylase on the apparent digestibility, ruminal fermentation and microbial protein synthesis of wethers as well as, the growth performance and meat quality of lambs. For that, two studies were carried out and in both studies the animals received one of four treatments (diets): 1) Corn silage uninoculated and without amylase added to TMR; 2) Corn silage uninoculated and amylase added to TMR; 3) Corn silage inoculated with 1×105 CFU LP [MA 18/5U] and 1×105 CFU BS [AT553098] without amylase added to TMR; 4) Corn silage inoculated with 1×105 CFU LP [MA 18/5U] and 1×105 CFU BS [AT553098] and amylase added to TMR. The enzyme utilized was amylase at the rate of 2 g of the product / kg of dietary dry matter (DM) (602 dextrinizing unit (DU)/kg of dietary DM). Amylase supplementation on the diet containing uninoculated silage increased (P=0.045) dry matter (DM) intake of wethers compared with wethers fed uninoculated silage without amylase supplementation (1,311 vs. 1,066 g/d), but not differed from others treatments. The apparent digestibility of DM, OM, CP, NDF and GE increased (P<0.01) in wethers fed with inoculated silages or supplemented with amylase, without interaction among inoculants and amylase. Wethers fed diets containing uninoculated silage and supplemented with amylase showed higher propionic acid and lower acetic acid proportion, with low acetic:propionic acid ratio, consequently. Microbial N supply tended to be higher (P=0.097) in wethers fed uninoculated silage with amylase supplementation and inoculated silage without amylase (8.01; 8.05 g/d). However, no effect was verified on the efficiency of microbial N synthesis. In the second study, lambs fed inoculated silage had higher NDF intake (P=0.019) than lambs fed uninoculated silage (266.5 vs 245 g/d). Lambs fed inoculated silage had higher average daily gain (P=0.019) when compared with lambs fed uninoculated silages (232.5 vs. 211.5). The inoculation of silage increased (P<0.05) the content of saturated fatty acid (SFA) and decreased (P<0.05) the unsaturated fatty acid (UFA) (47.55 vs. 46.21% and 52.44 vs. 53.79%, respectively) and consequently decreased the UFA:SFA ratio. The amylase supplementation at moment of feeding trended (P<0.10) to decrease the values of PUFA:SFA ratio (0.14 vs. 0.16). The association of amylase in diets containing inoculated silage did not provided positive responses on the digestibility and microbial N supply of wethers and did not alter the carcass and meat quality of lambs. Inoculation of silage with L. plantarum and B. subtilis improved the average daily gain of lambs when was not associated with amylase supplementation.
CNPq: 141008/2014-8

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CAPES
O experimento foi conduzido para avaliar as perdas por gases e efluente, as características fermentativas e microbiologicas, a composição bromatológica, bem como estimar os parâmetros cinéticos da digestibilidade ruminal in situ de silagens de capim Panicum Maximum Jacq. cv. Tanzânia inoculada com aditivo microbiano-enzimático. Foram testados o feitos de emurchecimento e inoculante, obtendo quatro tratamentos: silagem sem emurchecimento sem inoculante, silagem sem emurchecimento com inoculante, silagem emurchecida sem inoculante e silagem emurchecida com inoculante e três tempo de abertura(40 e 80 dias de ensilagem) e tempo zero sendo a forragem, distribuídos em um delineamento fatorial 2x2x3 inteiramente casualizado com três repetições. Foi feito um corte de uniformização da forrageira, realizado na altura de 30 cm do nível do solo. O capim tanzânia foi ensilado com 45 dias de rebrotação após o corte a 30 cm do solo e a forragem foi ensilada em silos experimentais de 1,3 litros, dotados de uma válvula de sifão e com 600 g de areia adicionados no fundo para retenção dos efluentes. A abertura dos silos ocorreu após 40 e 80 dias. Os dados foram analisadas utilizando o procedimento GLIMMIX do SAS, versão 9.3. Os efeitos principais e interações foram definidos pelo teste-F da ANOVA, sendo o comando SLICEDIFF usado para separar interações significativas. A opção LSMEANS foi utilizada para gerar as médias individuais para cada efeito. Por tratar-se de um fator quantitativo (tempo de abertura), contrastes ortogonais foram utilizados para partição específica dos efeitos de tempo de abertura com umidade e tipo de inoculante. O emurchecimento aumentou o teor de matéria seca de 18,10 para 44,18, mantendo em uma faixa ideal para conservação, melhorou características bromatologicas. Ainda reduziram as perdas por efluente e gás e N-amoniacal das silagens. O uso de inoculante microbiano-enzimático alterou as taxas de perdas por gases e nitrogênio amoniacal, de forma mais evidente em silagens com baixo teor de matéria seca. O inoculante aumentou a contagem lactobacilos nas forragens, diminui a contagem final de levedura e a concentração de acido acético e álcool. O emurchecimento diminui a concentração de acido acético, butírico e lático. Através do emurchecimento e inoculação se obteve o melhor valor de pH (4,54). O emurchecimento da forragem pode ser recomendado como técnicas para ensilagem de capim Tanzânia, pois melhoram a qualidade da silagem ao diminuírem os teores de ácido butírico e acético. O uso de aditivo microbiano-enzimático aliado ao emurchecimento na ensilagem do capim tanzânia, apresentaram o menor valor de pH diminuição.
The experiment was conducted to evaluate losses and effluent gases, fermentation and Microbiological characteristics, chemical composition, and to evaluate the kinetic parameters of in situ ruminal digestibility of grass silages Panicum Maximum Jacq. cv. Tanzania inoculated with microbial-enzyme additive. The inoculant made from wilting and tested, obtaining four treatments: no wilted silage inoculant without silage inoculant with no wilted silage inoculant and wilted without wilted silage inoculant and three time with opening (40 and 80 days of ensiling) and zero time being fodder, distributed in a 2x2x3 factorial design randomized with three replications. One of regrowth forage, held at the height of 30 cm from the ground level was done. Tanzania The grass was ensiled with 45 days of regrowth after cutting 30 cm of soil and forage was ensiled in experimental silos of 1.3 liters, equipped with a valve and siphon with 600 g of sand in the bottom for added retention of effluent. The Silos were opened after 40 and 80 days. Data were analyzed using the SAS procedure GLIMMIX, version 9.3. The main effects and interactions were defined by the F-test of ANOVA, and the command used to separate SLICEDIFF significant interactions. The LSMEANS option was used to generate the individual averages for each effect. Because it is a quantitative factor (opening time), orthogonal contrasts were used to partition the effects of specific opening time with humidity and type of inoculant. Wilting increased the dry matter content of 18.10 to 44.18, keeping in an ideal range for conservation, improved qualitative characteristics. Further reduced effluent losses and gas and ammonia-N silages. The use of microbial-enzyme inoculant shifted exchange losses gases and ammonia nitrogen, most clearly in silages with low content of dry matter. The inoculant increased lactobacilli count in fodder, reduces the final count of yeast and the concentration of acetic acid and alcohol. Wilting reduces the concentration of acetic, butyric and lactic acid. Through wilting and inoculation we got the best pH value (4.54). Wilting the forage can be recommended as techniques for ensiling Tanzania, for improving silage quality by decreasing the levels of butyric and acetic acid. The use of microbial-enzymatic additive combined with wilted grass silage of Tanzania, had the lowest pH value decreased.

Orientador: Ricardo Andrade Reis
Coorientador: Juliana Duarte Messana
Banca: Marcia Helena Machado da Rocha Fernandes
Banca: Giovani Fiorentini
Banca: Thiago Fernandes Bernardes
Banca: Odilon Gomes Pereira
Resumo: Objetivou-se neste trabalho avaliar os efeitos de dietas contendo silagem inoculada com Lactobacillus plantarum e Bacillus subtilis e suplementadas ou não com amilase sobre a digestibilidade aparente, fermentação ruminal e síntese de proteína microbiana em carneiros, assim como o desempenho e qualidade de carne de cordeiros. Para tanto, dois estudos foram conduzidos, no quais os animais receberam um dos quatro tratamentos (dietas): 1) silagem de milho não inoculada sem adição de amilase na mistura total da ração (MTR); 2) silagem de milho não inoculada e amilase adicionada na MRT; 3) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis, sem adição de amilase; 4) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis e amilase adicionada na MRT. A enzima utilizada foi a amilase numa taxa de aplicação de 2 g de produto / kg de matéria seca (MS) da dieta (602 unidade dextrinizante (UD) / kg de MS da dieta). A suplementação com amilase em dietas contendo silagem não inoculada aumentou (P=0,045) o consumo de matéria seca dos carneiros quando comparados com aqueles alimentados com silagem não inoculada sem suplementação com amilase (1,311 vs. 1,066 g/d), mas não diferiu dos outros tratamentos. A digestibilidade aparente da MS, MO, PB, FDN e EB aumentou (P<0,01) nos carneiros alimentos com silagem inoculada ou suplementados com amilase, sem interações entre os tratamentos. Os animais alimentados com dietas contendo ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Objetivou-se neste trabalho avaliar os efeitos de dietas contendo silagem inoculada com Lactobacillus plantarum e Bacillus subtilis e suplementadas ou não com amilase sobre a digestibilidade aparente, fermentação ruminal e síntese de proteína microbiana em carneiros, assim como o desempenho e qualidade de carne de cordeiros. Para tanto, dois estudos foram conduzidos, no quais os animais receberam um dos quatro tratamentos (dietas): 1) silagem de milho não inoculada sem adição de amilase na mistura total da ração (MTR); 2) silagem de milho não inoculada e amilase adicionada na MRT; 3) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis, sem adição de amilase; 4) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis e amilase adicionada na MRT. A enzima utilizada foi a amilase numa taxa de aplicação de 2 g de produto / kg de matéria seca (MS) da dieta (602 unidade dextrinizante (UD) / kg de MS da dieta). A suplementação com amilase em dietas contendo silagem não inoculada aumentou (P=0,045) o consumo de matéria seca dos carneiros quando comparados com aqueles alimentados com silagem não inoculada sem suplementação com amilase (1,311 vs. 1,066 g/d), mas não diferiu dos outros tratamentos. A digestibilidade aparente da MS, MO, PB, FDN e EB aumentou (P<0,01) nos carneiros alimentos com silagem inoculada ou suplementados com amilase, sem interações entre os tratamentos. Os animais alimentados com dietas contendo silagem não inoculada e suplementados com amilase apresentaram alta proporção de ácido propiônico e baixa de ácido acético, e consequentemente baixa relação de aceitoc:propiônico. A síntese de proteína microbiana tendeu a ser maior (P=0,097) nos carneiros alimentados com silagem não inoculada e suplementados com a... (Complete abstract click electronic access below)
Doutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Three thermophilic lignocellulolytic microbial communities were selected by enriched method with continual subcultivation at 55 °C. One community was selected from cow manure compost, another from decaying sugar cane bagasse, and the last one was obtained by mixing aliquots of the culture suspension from the first two. Those communities were able to degrade a cellulosic strip in three cultivation days. Evaluating the maximal day of CMCase and xylanase activity, it was observed that the mixed consortium showed the best results, with CMCase activity of 0.09 U mg-1 at the second day of cultivation, and xylanase activity of 2.86 U mg-1 at the fourth day. These enzymes were partially characterized with relation to temperature and pH of optimal activity. It was observed that CMCase showed the highest activity at 60 °C and pH 5.4, and kept 80% of its activity in a pH range of 4.5-6.5. On the other hand, the best activity for xylanase was verified at 65 °C, and in that same pH range, the enzyme kept 97% of its residual activity. Cell-free extract was concentrated by ultrafiltration. The enzymatic cocktail obtained showed CMCase activity 25-fold higher and xylanase activity 55-fold higher than the crude enzymatic extract. The cocktail was conserved by adding 50% glycerol. After storage for 60 days at 4 °C, xylanase kept 80% of the initial activity and CMCase didn t show loss of activity when kept at 25 °C for the same period. The mixed community cellular mass constitutes an inoculant able to maintain the cellulolytic phenotype after rapid freezing and storage at - 80 °C for 60 days. Fed-batch essay suggested that this community has potential to be manipulated in order to continuously hold the cellulolytic enzymes expression over time. The results evidenced the acquisition of an enzymatic cocktail from an inoculant which cellulolytic activity supported pH changes and optimal activity around 60 °C.
Três comunidades microbianas lignocelulolíticas e termofílicas foram selecionadas por subcultivo em meio de enriquecimento a 55 °C. Uma comunidade foi selecionada a partir de esterco de gado em compostagem, outra de bagaço de cana em decomposição e a terceira foi obtida a partir da mistura de alíquotas do meio de cultivo das duas anteriores. Tais comunidades apresentavam a característica de decompor uma fita de celulose em três dias de cultivo. Ao se avaliar o dia de máxima atividade CMCase e xilanase em cada comunidade, verificou-se que os melhores resultados foram encontrados no consórcio misto, com atividade de 0,09 U mg-1 para CMCase no segundo dia de cultivo e 2,86 U mg-1 para xilanase no quarto dia. Essas enzimas foram parcialmente caracterizadas em relação à temperatura e pH ótimos de atuação. Verificou-se que CMCase apresentou maior atividade a 60 °C e pH 5,4, e manteve 80% de sua atividade numa faixa de pH de 4,5 a 6,5. Já para a xilanase, a temperatura ótima foi de 65 °C e nessa mesma faixa de pH manteve uma atividade residual de 97%. O extrato livre de células foi concentrado por ultrafiltração e obteve-se um coquetel enzimático com atividade de CMCase e xilanase maior que no extrato bruto cerca de 25 e 55 vezes, respectivamente. O coquetel foi conservado pela adição de 50% de glicerol. Após 60 dias de armazenamento a 4 °C, a xilanase manteve 80% de sua atividade inicial e a CMCase não apresentou perda de atividade quando mantida a 25 °C pelo mesmo período. A massa celular da comunidade mista constitui um inoculante capaz de manter o fenótipo celulolítico após congelamento rápido e armazenamento a - 80 °C por 60 dias. Ensaio em batelada alimentada mostrou que essa comunidade apresenta potencial para ser manipulada a fim de se manter continuamente a expressão de enzimas celulolíticas ao longo do tempo. Os resultados mostraram que foi possível obter um coquetel de enzimas a partir do inoculante, cuja atividade celulolítica tolerou variações de pH e temperatura ótima em torno de 60 °C.

No contexto do desmatamento ocorrido ao longo dos anos no Estado do Paraná com a perda drástica da cobertura vegetal nativa e a crescente demanda por projetos de recuperação da mata ciliar, o estudo do Schizolobium parahyba (guapuruvú) para avaliar o seu potencial como fonte de madeira e de cobertura vegetal incrementar seu uso por ser uma espécie arbórea nativa da mata atlântica de rápido crescimento. O objetivo deste trabalho é o desenvolvimento de inoculantes microbianos que promovam o crescimento do guapuruvú em substituição ao adubo químico. Como inoculantes foram utilizados 10 g de inóculo bruto do fungo micorrízico arbuscular (FMA) Glomus clarum e como fixador de N, 1 mL de suspensão de 107 UFC de uma cepa de Rhizobium sp. No controle positivo foi aplicado 100 g por cova de adubo químico formulado NPK 20-5-20. O experimento foi conduzido em condição de campo, na estação experimental do IAPAR no município de Xambrê, PR. O delineamento experimental foi de blocos completos casualizados com cinco repetições e oito tratamentos (n = 800). A altura das plantas foi avaliada aos 30, 60, 120 180 e 240 dias. Os dados foram submetidos à ANOVA e as médias comparadas pelo teste de Tukey (p<0,05). Aos 30 dias as plantas não inoculadas com fungo MA apresentaram um maior crescimento quando comparadas com as inoculadas. O rizóbio e a micorriza não apresentaram efeito no crescimento das plantas. A alta concentração de P presente no solo pode ter influenciado negativamente o estabelecimento da simbiose com o FMA. Aos 60, 120, 180 e 240 dias, o guapuruvú respondeu significativamente ao tratamento com adubo em comparação com as plantas inoculadas. A sobrevivência foi maior no tratamento com adubo. Estes resultados demonstram que nos estádios iniciais a inoculação com microrganismos mostra-se pouco eficiente principalmente em plantas arbóreas nativas do grupo sucessional das pioneiras que apresentam uma alta taxa de crescimento como é o caso do guapuruvú quando cultivada em área de pastagem com Brachiaria decumbens.
In the context of deforestation occurred in the last decades in the Paraná state, where occurred a drastic loss of native vegetation, and now increasing demand a project of restoration of riparian forest. Schizolobium parahyba (guapuruvu) has a potential to be used as a source of wood and revegetation progam. S. parahyba is a native tree from Atlantic forest, that grow fast. The aim of this research was to develop microbial inoculants that promote the growth of guapuruvu replacing a chemical fertilizer. The plants were inoculated with 10 g of arbuscular mycorrhizal fungi (AMF) Glomus clarum and 1 mL of cell suspension (107 CFU/mL) of Rhizobium sp. and was considered as positive control 100 g of chemical fertilizer formula NPK 20-05-20 per plant. The experiment was carried out in field conditions, at the experimental station of IAPAR in municipality of Xambre, PR, Brazil. The experimental design was randomized complete blocks with five replications and eight treatments (n = 800). Plant height was measured at 30, 60, 120 180 and 240 days after planted. The results were analyzed by ANOVA and the Tukey test (p <0.05). At 30 days the plants not inoculated with AM fungi showed a higher growth when compared with inoculated. The rhizobia and mycorrhiza showed no effect on plant growth. The high concentration of P in soil may have negatively influenced the establishment of symbiosis with AMF. At 60, 120, 180 and 240 days, the fertilizer treatment increased plant growth when compared with inoculated plants. Survival was highest in the fertilizer treatment. These results demonstrated that in the early stages of growth, inoculation with microorganisms appeared to be not efficient especially in native woody plants of the successional group of pioneers that have a high rate of growth like the guapuruvu when cultivated in pasture area with Brachiaria decumbens.

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The aim of this study was to evaluate microbial populations, pH, the ammonium nitrogen/total nitrogen ratio, dry matter content, crude protein content, and the concentrations of acetic, propyonic, and butyric acids in sorghum and corn silage whether treated or not with two types of bacterial inoculants with only homo-lactic and hetero/homo-lactic strains in 20-L experimental silos. A 2 × 3 factorial scheme (two silages x three inoculants) was adopted in a complete random design whit three repetitions. An effect of the interaction silage versus inoculants was observed for all the studied variables. The acid lactic bacteria populations were recorded at 6.5 and 6.2 log cfu/g for the corn and sorghum plants, respectively, before ensilage. For the ammonium nitrogen/total nitrogen ratio, a lower value was observed for the sorghum silage treated whit homo-lactic strains, and a higher value of crude protein was also noted in this silage. Higher content of acetic acid was observed for the corn silage treated with hetero/homo-lactic inoculants. Regardless of the inoculants used, lower pH values were observed for the corn silage. Considering the evaluated traits, all the corn and sorghum silages can be rated as having good quality.
O objetivo deste estudo foi avaliar os teores de matéria seca, a relação nitrogênio amoniacal/nitrogênio total, o pH, os teores de proteína bruta, as populações microbianas e as concentrações dos ácidos acético, propiônico e butírico em silagens de milho e sorgo tratadas ou não com inoculantes bacterianos, contendo cepas homoláticas ou homo/heteroláticas. Foram utiliza- dos silos experimentais de 20 litros de capacidade, em um esquema fatorial 2 × 3 (dois tipos de silagem × três inoculantes), em delineamento inteiramente casualizado, com três repetições. Observou-se o efeito da interação silagem versus inoculante para todas as variáveis avaliadas. Constatou-se que o teor de matéria seca da silagem de milho não foi influenciado (P > 0,05) pelos inoculantes. No entanto, para a silagem de sorgo, verificou-se maior valor (P < 0,05) naquela tratada com cepas homoláticas. A relação N-NH3/N foi menor na silagem de sorgo tratada com inoculante homolático. Independentemente do inoculante usado, observou-se pH mais baixo na silagem de milho. Nas plantas de milho e sorgo, antes da ensilagem, foram registradas populações de bactérias láticas de 6,5 e 6,2 log ufc/g, enquanto nas silagens estas populações foram da ordem de log 7 e log 6, respectivamente. A contagem de fungos e leveduras foi maior na silagem de milho. Foi observado maior teor de ácido acético na silagem de milho tratada com inoculante heterolático. Considerando as características avaliadas, as silagens de milho e de sorgo, tratadas ou não com inoculantes bacterianos com cepas homoláticas ou homo/heteroláticas, podem ser consideradas de boa qualidade.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
A presente dissertação foi elaborada com base em dois experimentos. No primeiro, avaliou-se a utilização de inoculante microbiano em relação a melhora no consumo voluntário e na digestão dos nutrientes das dietas, e avaliar a substituição do milho pelo sorgo em relação ao desempenho, e sobre as alterações sensoriais na carne de cordeiros terminados em confinamento. Foram utilizados 28 cordeiros machos não castrados F1 Dorper × Santa Inês, com peso corporal médio inicial de 20,0±0,7 kg e idade entre três e quatro meses. Inicialmente, quatro animais foram abatidos e tomados como referência para estimar o peso de corpo vazio (PCVZ) inicial dos animais remanescentes no experimento. Os demais 24 animais foram distribuídos em delineamento inteiramente casualizado em esquema fatorial 2 × 2, e cada grupo foi alimentado com uma das dietas composta por dois tipos de grãos ensilados e reidratados: milho (MR) ou sorgo (SR), adicionados (I) ou não de inoculante microbiano (composto de Lactobacillus plantarum, Propionibacterium acidipropionici e sacarose), com seis repetições. Os grãos de milho e sorgo foram reidratados para 65% de matéria seca, sendo ensilados em toneis plásticos. O volumoso foi a silagem de milho. O experimento foi dividido em três períodos de 28 dias. As digestibilidades das dietas foram avaliadas dos 35o aos 40o dias e 75° aos 80o dias do período experimental, utilizando-se a fibra em detergente neutro indigestível (FDNi) para estimar a excreção fecal. Ao final dos 84 dias, todos os animais foram abatidos. Não houve interação (P > 0,05) entre tipo de grão e inoculante para nenhumas das variáveis avaliadas. Os consumos de matéria seca (MS), e dos nutrientes assim como dos nutrientes digestíveis totais (NDT) não foram influenciados pelo tipo de grão (milho ou sorgo) ou pela presença de inoculante (P > 0,05). Os coeficientes de digestibilidade para MS e demais nutrientes, também não foram alterados (P > 0,05) pelo tipo de grão ou pela inclusão de inoculante microbiano. O ganho médio diário (GMD), o rendimento de carcaça (RC), a eficiência alimentar (EA) e os atributos qualitativos da carne como perdas por cocção (PCc), descongelamento (PD) e total (PT), maciez (FC), pH, acabamento de gordura (EGS) e cor, também não foram influenciados pelo tipo de grão ou pela presença do inoculante microbiano (P > 0,05). Conclui-se que as dietas contendo silagem de grãos reidratados de milho ou sorgo foram igualmente eficientes para a terminação de ovinos e que o inoculante microbiano utilizado não foi capaz de promover melhorias no desempenho produtivo de ovinos. No segundo experimento, avaliou-se utilização de inoculante microbiano, o processamento na forma de reidratação e ensilagem dos grãos de milho e sorgo em relação à melhora da digestão do amido e digestão dos nutrientes das dietas, no crescimento microbiano ruminal e a retenção de nitrogênio no organismo do animal. Foram utilizados cinco ovinos não castrados com peso médio inicial de 30,0±2,4 kg distribuídos em um delineamento em quadrado latino 5 × 5, em esquema fatorial 2 × 2 + 1, constituídos por dois tipos de grãos reidratados: milho (MR) ou sorgo (SR), adicionados (I) ou não de inoculante microbiano e um tratamento contendo milho moído (M). Os grãos de milho e sorgo no tratamento de reidratação alcançou 65% de matéria seca, sendo ensilados em toneis plásticos. O volumoso utilizado foi a silagem de milho. O experimento teve a duração de 105 dias, dividido em cinco períodos de 21 dias cada, sendo 14 dias para adaptação e sete dias para coleta de amostras. Houve interação (P < 0,05) entre tipos de grãos e uso de inoculante microbiano apenas para a digestibilidade do amido, dentre as variáveis testadas. O consumo de MS, dos demais nutrientes e dos nutrientes digestíveis totais (NDT), assim como os coeficientes de digestibilidade não foram influenciados pela interação (P > 0,05) tipo de grão (milho ou sorgo) e adição ou não de inoculante. Adicionalmente, não se observaram efeito (P > xi0,05) do tipo de grão, inoculante (P > 0,05) e do tipo de processamento (M vs grãos de milho e sorgo reidratados (GR; P > 0,05). Entretanto, a digestibilidade do amido, apresentou interação (P < 0,05) entre tipo entre tipos de grãos e uso de inoculante microbiano, e também, foram influenciados (P < 0,05) pelo tipo de grão (milho ou sorgo), inclusão de inoculante e tipo de processamento (seco ou reidratado), sendo maior a digestibilidade do amido dos grãos que passaram pelo processamento de reidratação e uso de inoculante. Não houve efeito de interação entre tempo e dietas experimentais (P > 0,05) sobre os valores de pH no fluido ruminal; no entanto, observou-se efeito de tempo sobre esta variável (P < 0,05). Adicionalmente, não se observaram efeitos de interação entre tipo de grão e inoculante (P > 0,05), efeito do tipo de grão (P > 0,05), inoculante e do tipo de processamento (M vs GR). Não houve efeito de interação entre tempo e dietas experimentais (P > 0,05) sobre as concentrações de N-NH 3 no fluido ruminal; no entanto, observou-se efeito de tempo sobre esta variável (P < 0,05). Adicionalmente, observou-se efeitos (P < 0,05) para o tipo de processamento (M vs GR), em que os grãos reidratados obtiveram maiores médias. O balanço de nitrogênio e eficiência microbiana não foram influenciados pela interação (P > 0,05) tipos de grãos e adição ou não de inoculante. Também, não se observou efeito (P > 0,05) sobre tipo de grão, inoculante (P > 0,05) e do tipo de processamentos (M vs GR; P > 0,05). Conclui-se que dietas contendo grãos processados na forma seca ou reconstituída foram igualmente eficientes no funcionamento do rúmen, que o tipo de grãos também não altera os parâmetros ruminais e que o inoculante microbiano utilizado não foi capaz de promover melhorias na digestibilidade das dieta de ovinos.
The present dissertation was elaborated based on two experiments. In the first one was evaluated the effect of the microbial inoculant and the substitution of maize by sorghum on voluntary consumption and nutrient digestion of diets, performance, and sensorial changes in the meat of lambs finished in confinement. Were used 28 male lambs, not castrated F1 Dorper × Santa Inês, with initial body weight of 20,0 ± 0,7 kg, and age between three and four months. Initially, four animals were slaughtered and taken as reference to estimate the initial empty body weight (EBW) of the animals remaining in the experiment. The remaining 24 animals were distributed in a completely randomized design in a 2 × 2 factorial scheme, and each group was fed with one of the diets composed of two types of grains ensiled and rehydrated corn (RC) or sorghum (RS), added (A) or not of inoculant microbial (compound of Lactobacillus plantarum, Propionibacterium acidipropionici and sucrose), with six replicates. The corn and sorghum grains were rehydrated to 65% of dry matter, being ensiled in plastic sherds. The volume was corn silage. The experiment was divided into three periods of 28 days. The digestibilities of the diets were evaluated from the 35 th to the 40 th day, and from the 75th to the 80th day of the experimental period, using the indigestible neutral detergent fiber (INDF) to estimate fecal excretion. At the end of the 84 days, all animals were slaughtered. There was no interaction (P > 0,05) between grain type and inoculant for any of the evaluated variables. Consumption of dry matter (DM) and nutrients as well as total digestible nutrients (TDN) were not influenced by the type of grain (corn or sorghum) or by the presence of inoculant (P > 0,05). The digestibility coefficients for DM and other nutrients were also not altered (P > 0,05) by grain type or by the inclusion of microbial inoculant. The average daily gain (ADG), carcass yield (CY), feed efficiency (FE) and the qualitative attributes of the meat as cooking losses (CL), thawing (TL) and total, softness (FC), pH, fat finishing (FF) and color were also not influenced by the type of grain or by the presence of the microbial inoculant (P > 0,05). It was concluded that the diets containing rehydrated grain silage of maize or sorghum were equally efficient for the finishing of sheep and that the microbial inoculant used was not able to promote improvements in the productive performance of sheep. In the second experiment, the use of microbial inoculant was evaluated in the processing, in the rehydration and ensilage of maize and sorghum grains and on the digestion of the starch and other nutrients of the diets, microbial growth and retention of nitrogen in the animal's organisms.Were used five uncastrated lambs with an initial average weight of 30.0 ± 2.4 kg distributed in a 5 × 5 Latin square design, in a 2 × 2 + 1 factorial scheme, consisting of two types of rehydrated grains corn (RC) or sorghum (RS), added (A) or not of microbial inoculant and a treatment containing ground corn (C). The maize and sorghum grains in the rehydration treatment reached 65% dry matter, being ensiled in plastic barrels. The volume used was corn silage. The experiment lasted 105 days, divided into five periods of 21 days each, being 14 days for adaptation and seven days for sample collection. There was interaction (P < 0,05) between grain types and use of microbial inoculant only for starch digestibility, among the variables tested. Consumption of (DM), of others dietary constituents and total digestible nutrients (TDN), as well as the digestibility coefficients were not influenced by the type of grain (corn or sorghum) interaction (P > 0,05) and addition or absence of inoculant. In addition, no effect (P > 0,05) of the grain type was observed, and the type of processing (dry corn grains vs rehydrated corn and sorghum grains (RG; P > 0,05) However, the digestibility of the starch presented interaction (P < 0.05) between grain types and the use of microbial inoculant, and was also influenced by xivthe (P < 0,05) type of processing (dry or rehydrated), being greater the digestibility of starch obtained for the grains that went through the processing of rehydration and use of inoculant. There was no interaction effect between time and experimental diets (P > 0,05) on pH values in the ruminal fluid however, we observed a time effect on this variable (P < 0,05). In addition, no interaction effects were observed between grain type and inoculant (P> 0.05), effect of grain type (P > 0,05), inoculant and type of processing (C vs RG). There was no interaction effect between time and experimental diets (P > 0,05) on the concentrations of N-NH3 in the ruminal fluid. However, we observed a time effect on this variable (P < 0,05). In addition, an effect (P < 0,05) was observed for the type of processing (C vs RG), in which the rehydrated grains obtained higher averages. Nitrogen balance and microbial efficiency were not influenced by the interaction (P > 0,05), grain types and addition or not of inoculant. Also, no effect (P > 0,05) was observed on grain type, inoculant (P > 0,05) and type of processing (C vs RG; P > 0,05). It is concluded that diets containing grains processed in dry or reconstituted form were equally efficient in rumen function, that the type of grains also does not alter the ruminal parameters, and that the microbial inoculant used was not able to promote improvements in the digestibility of sheep diets.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Avaliou-se o perfil fermentativo, a composição química e as populações microbianas de silagens de alfafa emurchecida ou não, tratadas com inoculante bacteriano em diferentes períodos de fermentação; bem como o isolamento, a identificação e a caracterização das bactérias do ácido lático (BAL) das silagens não inoculadas. Foi utilizado um esquema fatorial 2 × 2 × 6, ausência ou presença de emurchecimento (E), com e sem inoculante bacteriano (I) e seis períodos de fermentação (P) (1, 3, 7, 14, 28 e 56 dias), no delineamento inteiramente casualizado, com três repetições. A alfafa foi colhida aos 50 dias de rebrotação e picada em partículas de 1,5 cm. A alfafa foi emurchecida por 6h, no campo, no material submetido a esse tratamento. O inoculante usado foi o Sil-All® 4x4 W.S. (Alltech, Brasil), aplicado na taxa de 10 5 UFC/g (5 g/t de forragem). Após a aplicação dos tratamentos, a forragem foi ensilada em sacos plásticos de 25 × 35 cm e selados a vácuo. O emurchecimento da alfafa reduziu a capacidade tampão e as concentrações de carboidratos solúveis em água e aumentou o teor de MS da forragem de 133,9 g/kg MS para 233,4 g/kg MS, antes da ensilagem. Foi observado efeito (P < 0,05) de E sobre os teores de MS, PB, NIDA e FDN das silagens aos 56 dias de fermentação. As populações de BAL e fungos não foram afetadas por nenhum dos fatores estudados, enquanto as populações de enterobactérias e leveduras foram afetadas (P < 0,05) pelo P. O pH e a concentração de nitrogênio amoniacal foram afetados (P < 0,05) pela interação E × P, registrando-se menores valores destas variáveis nas silagens emurchecidas. Também foi observado efeito (P < 0,05) da interação E × P sobre o conteúdo dos ácidos lático, acético e propiônico, sendo registrados menores valores de ácido acético nas silagens emurchecidas em todos os períodos de fermentação. Menores concentrações de ácido propiônico também foram encontradas nas silagens emurchecidas. Excetuando o 1o dia de fermentação, foram observadas maiores concentrações de ácido lático nas silagens emurchecidas, no entanto, a partir do 7o dia, a concentração deste ácido decresceu em ambas as silagens emurchecidas ou não. A adição de inoculante bacteriano na presença ou não do emurchecimento, não promove melhorias no x processo fermentativo de silagem de alfafa com elevada umidade. A população de BAL na planta e nas silagens de alfafa submetidas ou não ao emurchecimento é diversa em nível de espécies, o Lactobacillus plantarum é a espécie predominante e apresenta comportamento distinto ao longo dos períodos de fermentação nas silagens não emurchecidas e emurchecidas.
We aim with this study to evaluate the fermentation characteristics, chemical composition and microbial populations of wilted or unwilted alfalfa silage treated with bacterial inoculant in different periods of fermentation as well as the isolation, identification and characterization of lactic acid bacteria (LAB) from non-inoculated silage. We analyzed the data as a 2 × 2 × 6 factorial arrangement with the effects of wilting (W) (absence or presence), bacterial inoculant (I) (with and without) and period (P) (1, 3, 7, 14, 28 and 56 days), and their interaction in a completely randomized design with three replications. We harvested the alfalfa at 50 days of regrowth, chopped into particles of 1.5 cm and wilted for 6 hours in the field before ensiling. The commercial inoculant used was Sil-All® 4x4 W.S. (Alltech, Brazil), applied at the rate of 10 5 CFU/g of fresh forage. After the treatments forage was packed in plastic bags of 25 × 35 cm and sealed by using vacuum sealer. Wilting of alfalfa reduced buffering capacity and water soluble carbohydrate concentrations and increased the dry matter (DM) content of the forage 133.9 g/kg DM to 233.4 g/kg DM before ensiling. There was effect (P < 0.05) of W on the DM, crude protein, neutral detergent fiber and acid detergent insoluble nitrogen in the silages at 56 d. Populations of LAB and molds were not affected by any of the effects studied, however the populations of enterobacteria and yeasts were affected (P < 0.05) by P. The pH and ammonia nitrogen concentration were affected by the interaction W × P (P < 0.05), registering lower values of these variables on wilted silages. There was effect (P < 0.05) of the interaction W × P on the concentrations of lactic, acetic and propionic acid, resulting in lower values of acetic acid in the wilted silages in all fermentation periods. Lower concentrations of propionic acid were found in wilted silages. Except for the 1st day of fermentation, we observed higher concentrations of lactic acid in the wilted silages, however in both wilted and unwilted silages the values decreased from the 7th d of ensiling. The addition of inoculant in presence or absence of wilting, does not improve the fermentation process of alfalfa silage with high moisture content. The populations of LAB in the wilted or unwilted fresh-alfalfa and in the silages are diverse in species levels and the xii Lactobacillus plantarum is the predominant specie and presents different dynamic appearance over the fermentation period between unwilted and wilted silages.

Resumo: Objetivou-se avaliar a estabilidade aeróbia de silagens de planta e de grãos úmidos de milho com a utilização de inoculante microbiano, bem como o uso de filme plástico de baixa permeabilidade ao oxigênio associado ao filme convencional. Para tanto foram conduzidos três experimentos. No primeiro e segundo experimentos os objetivos foram avaliar os efeitos de doses de Lactobacillus buchneri (5x104, 1x105, 5x105 e 1x106 UFC/ g) sobre as características fermentativas, a dinâmica microbiológica, a estabilidade aeróbia e o valor nutricional de silagens de planta e de grãos úmidos de milho. No terceiro experimento, objetivou-se avaliar o efeito do filme plástico de baixa permeabilidade ao oxigênio associado ao filme convencional sobre a perdas na armazenagem e a estabilidade aeróbia de silagem de planta de milho, em silo tipo superfície. O L. buchneri é eficiente no controle de leveduras e fungos filamentosos, assim como melhora a estabilidade aeróbia da silagem de planta e de grãos úmidos de milho e não afeta o valor nutricional. O filme de baixa permeabilidade ao oxigênio associado ao filme convencional é eficiente na manutenção das características fermentativas, no controle da população de leveduras e na redução de perdas, principalmente quando a fatia de retirada é menor
Abstract: Aimed to evaluate the aerobic stability of plant and of high moisture corn silage with the use of microbial inoculant and oxygen barrier film associated with conventional film. Therefore, three experiments were conducted. In the first and second experiments the objectives were to evaluate the effects of Lactobacillus buchneri doses (5x104, 1x105, 5x105 and 1x106 CFU/g) on the fermentation characteristics, microbial dynamics, aerobic stability and nutritional value of plant and of grain moisture corn silage. The third experiment aimed to evaluate the effect of oxygen barrier film associated with conventional film under the storage losses and aerobic stability of plant corn silage in a horizontal silo. The L. buchneri is effective in control of yeasts and molds, and improve the aerobic stability of plant and of high moisture corn silage and do not affect nutritional value. The oxygen barrier film is effective in maintaining the fermentation characteristics, in yeasts population control and loss reduction, mainly when the feedout rate is lower
Orientador: Ricardo Andrade Reis
Coorientador: Thiago Fernandes Bernardes
Coorientador: Ana Claudia Ruggieri
Banca: Clóves Cabreira Jobim
Banca: Antonio Fernando Bergamaschine
Mestre

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O presente trabalho teve o objetivo de avaliar técnicas, como o uso de aditivos microbianos direcionados ao controle de microrganismos aeróbios, bem como a utilização de um filme plástico de baixa permeabilidade ao oxigênio no controle da deterioração aeróbia. No primeiro estudo, o objetivo foi avaliar o efeito do Lactobacillus buchneri sobre a composição química, o perfil de fermentação, a presença de fungos e de leveduras e a estabilidade aeróbia da silagem de milho. No segundo, objetivou-se avaliar as perdas de matéria seca, a composição químico-bromatológicas e a presença de fungos e leveduras das silagens de milho, na região periférica de silos superfícies, quando vedada com filme de baixa permeabilidade ao oxigênio. A inoculação influenciou o perfil fermentativo, indicando que o aumento da dose aplicada na massa ensilada reduziu a recuperação da MS e aumentou as perdas por gases. A composição bromatológica das silagens de milho não foi influenciada, no entanto ocorreu maior estabilidade aeróbia das silagens tratadas. O filme de baixa permeabilidade ao oxigênio reduz a ocorrência de leveduras e de fungos quando é imposto ao silo um avanço satisfatório da massa de silagem durante o desabastecimento, o que poderia melhorar a qualidade sanitária da mesma.
The present work aimed to evaluate techniques, as additives specific for aerobic microorganisms control and plastic film with low oxygen permeability for aerobic deterioration control. In the first study, the aim was to evaluate the effect of Lactobacillus buchneri on the chemical composition, the fermentation profile, the presence of fungi and yeasts and aerobic stability of corn silage. In the second, it was aimed to evaluate the dry matter losses, chemical-bromatologic compound and the presence of fungi and yeasts of corn silage, in the peripheral region of surfaces silos when forbidden to film from low permeability to oxygen. The inoculation influenced the fermentation profile, indicating that the increase of the dose applied in mass ensiled reduced the recovery of MS and increased losses by gases. The corn silage chemical composition was not influenced however there was greater aerobic stability of the treated silage. The low oxygen permeability film reduces the occurrence of yeast and fungi when it is imposed on a breakthrough satisfactory silo of the mass of silage during feedout rate, which could improve the health quality of the same.

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CAPES
One of the most known and important process in nature is the biological nitrogen fixation (FBN). Few classes of microorganisms perform this process; named diazotrophics that associated with plants is an important alternative to enhance nitrogen nutrition in agricultural systems. Diazotrophic bacterial population in association with sugarcane improves the yield due to its ability to colonize the inner roots of the host plant and do not provoke any pathogenicity signal. Quantitative analyzes have indicated that selection of combinations of endophytic diazotrophic strains when associated with sugarcane varieties can improve the yield and therefore varieties of sugarcane may be better exploited in order to benefit this association for agricultural purposes. Thus, this work proposes to monitor the establishment of diazotrophic bacteria present in the inoculant of Embrapa, during the initial development of sugarcane plants. Micro-propagated sets of the sugarcane variety RB867515 were inoculated individually and with a mixture of five nitrogen fixing bacteria strains that compose the sugarcane inoculant: Nitrospirillum amazonense (CBAmC) Paraburkholderia tropica (Ppe8) Gluconacetobacter diazotrophicus (Pal5) Herbaspirillum seropedicae (HRC54) and Herbaspirillum rubrisubalbicans (HCC103). Plants were grown in a greenhouse and harvested 30 days after planting. Fresh root material was separated for evaluation of the establishment of the strains by bacterial counting using the technique of Most Probable Number (MPN), absolute quantification by Real Time PCR (qPCR), besides the identification of the inoculated strains through analysis of the rDNA profile of the pellicle formed in the respective semisolid media using the BOX-PCR technique. The results of bacterial count diazotrophic present in the roots by NMP showed that the treatments inoculated in the control (native population) showed higher population of bacterial cells around 105 cells by fresh root mass, but not statistically significant. The methodology qPCR permitted quantitation of the number of 16S rDNA copies in the order of 105 bacterial cells g - 1 fresh weight root, showing that there were differences in the population of endophytic species that colonize the roots of sugarcane. The profiles of BOX -PCR of the respective pellicles formed in semisolid media did not show high similarity (> 80 %) with the profile of the species inoculated for most of the treatments. The results obtained indicate that the qPCR technique showed the establishment of some of the inoculated strains in sugar cane buds while the NMP technique showed no significant difference between treatments inoculated and non-inoculated possibly due to the lower sensitivity of the method.
Um dos processos mais conhecidos e importantes na natureza ? a fixa??o biol?gica de nitrog?nio atmosf?rico (FBN). Este processo ? realizado por algumas classes de microrganismos, denominados diazotr?ficos, que associados com esp?cies vegetais s?o uma alternativa importante para aprimorar a nutri??o nitrogenada nos sistemas agr?colas. As popula??es de bact?rias fixadoras de nitrog?nio atmosf?rico quando associadas ? cultura da cana-de-a??car melhoram a produ??o e, ao colonizarem o interior das ra?zes promovem benef?cios ?s plantas hospedeiras. An?lises quantitativas t?m indicado que a sele??o das combina??es de estirpes de bact?rias diazotr?ficas endof?ticas e variedades de cana-de-a??car podem ser melhor exploradas com o objetivo de aperfei?oar a associa??o para finalidades agr?colas. Assim, o presente trabalho prop?e monitorar o estabelecimento das bact?rias diazotr?ficas presentes no inoculante para cana-de- a??car da Embrapa, durante o desenvolvimento inicial planta. Toletes propagados da variedade RB867515 de cana-de-a??car foram inoculados individualmente e com a mistura das cinco estirpes de bact?rias diazotr?ficas: Nitrospirillum amazonense (CBAmC), Paraburkholderia tropica (PPe8), Gluconacetobacter diazotrophicus (Pal5), Herbaspirillum seropedicae (HRC54) e Herbaspirillum rubrisubalbicans (HCC103). As plantas foram crescidas em casa de vegeta??o e coletadas 30 dias ap?s o plantio, sendo separado o material vegetal para avalia??o do estabelecimento das estirpes pela t?cnica do N?mero Mais Prov?vel (NMP) e quantifica??o absoluta por PCR em Tempo Real (qPCR). Adicionalmente foi feita a identifica??o das estirpes inoculadas atrav?s da an?lise do perfil de rDNA das pel?culas bacterianas formadas nos respectivos meios semiss?lidos pela t?cnica de BOX-PCR. Os resultados obtidos da contagem de bact?rias diazotr?ficas presente nas ra?zes por NMP mostrou que os tratamentos inoculados em rela??o ao controle (popula??o nativa), apresentaram uma maior popula??o de c?lulas bacterianas em torno de 105 c?lulas por massa fresca de raiz, por?m n?o significativa. A metodologia de qPCR permitiu a quantifica??o do n?mero de c?pias do 16S rDNA, da ordem de 105 c?lulas bacterianas g-1 massa fresca de raiz, mostrando que houve diferen?a na popula??o das esp?cies diazotr?ficas endof?ticas que colonizam as ra?zes de cana-de-a??car. Os perfis obtidos por BOX-PCR das pel?culas formadas nos respectivos meios semiss?lidos n?o mostraram alta similaridade (>80%) com o perfil das esp?cies inoculadas para a maioria dos tratamentos inoculados. Os resultados obtidos indicam que a t?cnica de qPCR permitiu mostrar o estabelecimento de algumas estirpe do inoculante nos toletes de cana-de a??car enquanto que a t?cnica de NMP n?o mostrou diferen?a significativas entre os tratamentos inoculados e n?o inoculados possivelmente pela menor sensibilidade da metodologia.

A ensilagem da cana-de-açúcar sem o controle da produção de etanol resulta em silagens de baixa qualidade, podendo reduzir o consumo voluntário e o desempenho animal. O valor nutritivo de silagens de cana-de-açúcar e seus efeitos sobre o desempenho e o comportamento ingestivo de cabras em lactação e de cordeiros confinados foram avaliados em diferentes ensaios. Trinta e nove cabras da raça Saanen foram utilizadas para avaliar o consumo de matéria seca (CMS), a produção e composição do leite, os componentes sangüíneos e o comportamento ingestivo. Trinta cordeiros da raça Santa Inês foram confinados para avaliar o desempenho, as características da carcaça e o comportamento ingestivo. Os animais foram alimentados com rações compostas de 50% de volumoso e 50% de concentrado, diferindo quanto ao tipo do volumoso utilizado: cana-de-açúcar in natura, silagem de cana-de-açúcar sem aditivo e silagem de cana-de-açúcar aditivada com L. buchneri (5x104 ufc/g MV), constituindo os tratamentos experimentais SC, SCS e SCS+Lb, respectivamente. O comportamento ingestivo foi realizado individualmente, com observações feitas a cada cinco minutos. Amostras das silagens foram analisadas para matéria seca (MS), proteína bruta (PB), fibra em detergente neutro (FDN), fibra em detergente ácido (FDA), carboidratos solúveis (CHOS), ácido acético e etanol, e as médias foram comparadas com a cana-de-açúcar in natura. A estabilidade aeróbia foi avaliada através do controle da temperatura, pH e perdas de MS das silagens expostas ao ar. Para determinar os coeficientes de digestibilidade e o balanço de nitrogênio das rações utilizadas na avaliação de desempenho, 12 borregos foram mantidos em gaiolas para ensaios de metabolismo. O CMS foi maior (P<0,01) para as cabras que receberam o tratamento SC. Não houve diferença (P>0,05) para a produção de leite e produção de leite corrigida para 3,5% de gordura. Os teores de gordura e de sólidos totais no leite foram maiores para as dietas contendo silagem, sendo superior (P<0,01) para o tratamento SCS+Lb. A concentração plasmática de ácidos graxos não esterificados (AGNE) e o tempo despendido com ruminação (min/g MS) foi maior (P<0,05) para os animais alimentados com as silagens. Não houve diferença (P>0,05) para o CMS, ganho de peso vivo, conversão alimentar e parâmetros de carcaça dos cordeiros. O tempo de ingestão (min/g FDN) e a eficiência de ruminação foram inferiores (P<0,05) nos cordeiros alimetados com silagem de cana-de-açúcar. A ensilagem da cana-de-açúcar resultou em redução (P<0,01) no teor de carboidratos solúveis e aumento (P<0,01) nas concentrações da FDN, FDA, hemicelulose e ácido acético. Os teores de MS, CHOS e ácido acético foram maiores (P<0,01) para o tratamento SCS+Lb. Não houve diferença (P>0,01) no teor de etanol entre as silagens. A silagem aditivada apresentou menores (P<0,05) perdas de MS, manutenção do pH e maior (P<0,05) estabilidade durante o período de avaliação em aerobiose. O consumo e a digestibilidade aparente da FDN, FDA e hemicelulose foram maiores (P<0,05) para a rações contendo silagem.
Sugar cane ensiled without controlling ethanol production results in low quality roughage and may decrease voluntary feed intake and animal performance. Sugar cane silages nutritive value and their effects on performance and ingestive behavior of lactating goats and feedlot lambs were evaluated in different trials. Thirty-nine Saanen does were used to evaluate dry matter intake (DMI), milk yield and composition, blood parameters and ingestive behavior. Thirty Santa Ines ram lambs were penned to evaluate performance, carcass characteristics and ingestive behavior. Animals were fed a 50:50 (concentrate:roughage ratio) total mixed ration, with different roughages: fresh sugar cane, sugarcane silage without additive and sugar cane silage treated with Lactobacillus buchneri (5x104 cfu/g wet basis) corresponding to the experimental treatments SC, SCS and SCS+Lb, respectively. Ingestive behavior was evaluated individually every 5 minutes for 24 h. Silages were sampled and analyzed for dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), soluble carbohydrates (CHOS), acetic acid and ethanol and compared with fresh sugar cane. Aerobic stability was evaluated by controlling the temperature, pH and dry matter losses of silages exposed to air for a period of ten days. To evaluate apparent digestibility and N metabolism of diets used in the performance trials, 12 ram lambs were placed in metabolism crates. DMI was higher (P<0.01) for goats fed SC. Milk production (MP) and fat corrected milk yield were similar (P>0.05). Milk fat and total solids were greater for diets containing silages. Does fed SCS+Lb diet had higher (P<0.01) milk fat content than SCS. NEFA concentration and time spent with rumination (min/g DM) was higher (P<0.05) for animals fed silage diets. There were no differences (P>0.05) on dry matter intake, average daily gain, feed conversion and carcass parameters of the feedlot lambs. Eating time (min/g NDF) and rumination efficiency (g DM/h) were lower (P<0.05) for lambs fed silage diets. Sugar cane silage decreased (P<0.01) soluble carbohydrates concentrations and increased (P<0.01) NDF, ADF, hemicelulose and acetic acid levels. DM, soluble carbohydrates and acetic acid were higher (P<0,01) for SCS+Lb. There was no difference (P>0.01) on ethanol levels between silages. SCS+Lb treatment had lower (P<0.01) dry matter losses, unchanged pH and greater (P<0.05) aerobic stability. NDF, ADF and hemicelulose intakes and apparent digestibility were higher (P<0.05) for silage treatments.

Tese no âmbito do Doutoramento em Biociências, ramo de especialização em Biotecnologia, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e tecnologia da Universidade de Coimbra.
The interest in plant growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal (AM) fungi for agricultural purposes (e.g. enhancement of crop yield and nutrition, promotion of plants resilience to abiotic stress) is rising. Yet, large-scale applications of these microbes have been hampered by the lack of data on their field performance and the feasibility of the inoculation methods, especially in the case of AM fungi. Seed coating, a technique in which seeds are covered with minor amounts of exogenous materials (including microbial inoculants), is a potential tool to deliver microbes at large-scale. This technique has been gaining attention in the agricultural sector. A literature review revealed that seed coating has been applied to more than 50 plant species (e.g. wheat, tomato, maize, melon, bean, clover), including seeds with different characteristics (dimensions, forms, textures). Mostly studied for the application of various species of PGPR (especially from the genera Pseudomonas and Bacillus), seed coating is not so frequently explored for inoculation of AM fungi or microbial consortia. The improvement of crop productivity and protection of plants against pathogens have been the main focus of research on microbial seed coating, while a smaller portion has been aimed at enhancing crops resistance to abiotic stresses. One of the main goals of this PhD thesis was to evaluate seed coating as a deliver system for the inocula of AM fungi and PGPR. Overall, seed coating allowed the application of minor amounts of AM fungi and PGPR to the seeds of three selected agricultural crops: maize, cowpea and chickpea. Further, by comparing inoculation of AM fungi through direct soil inoculation (conventional) with seed coating, similar AM root colonization was obtained despite a reduction in the amount of applied inocula. Contrary to AM fungi, the presence of PGPR coated on the seeds could not be confirmed in the rhizosphere and roots of the inoculated crops. It is well known that both AM fungi and PGPR have the ability to improve soil fertility and enhance plant nutrition, which can bring benefits for plant growth and development. By increasing nutrient availability and nutrient use efficiency, these plant beneficial microbes (PBM) can assist farmers to reduce their dependence on chemical fertilizers. The results obtained in this thesis showed that coating seeds with PGPR and AM fungi had a significant impact on plant shoot nutrient concentrations under different fertilization regimes. For instance, maize seeds coated with AM fungi (Rhizophagus irregularis) increased shoot nutrient concentration (nitrogen, phosphorus, potassium, magnesium and zinc), comparing with non-inoculated plants. Nutrient contents on maize shoot were boosted by R. irregularis inoculation, particularly in the treatments where fertilization was reduced or absent. On the contrary, maize coated with PGPR (Pseudomonas fluorescens) presented most of the nutritional increments when full fertilization was applied. Nevertheless, in both inoculation treatments, despite the nutrient enhancements, no improvement in plant biomass was obtained. These results confirm that PBM can increase plant nutrient uptake. PGPR and AM fungi are known to confer drought resistance to plants. Coating seeds with PGPR (Pseudomonas putida) showed a general positive influence in the plant productivity, especially under moderated water deficit. Seed coating with AM fungi (R. irregularis singly or in consortia with P. putida) promoted nutrient uptake, leaf pigment contents and gas exchange parameters of cowpea, yet mostly when plants where under no water deficit. Mainly, these results emphasized the importance of selecting the PBM that better potentiate plant resilience to abiotic stresses, in order to obtain the best benefits from the inoculation. Field experiments are essential to validate the benefits of microbial seed coating and its feasibility for large-scale applications. A comparison between chickpea coated with a single AM fungal isolate of R. irregularis and multiple isolates of the same fungal species under greenhouse and field conditions showed that plants inoculated with multiple AM fungal isolates performed better (e.g. higher biomass, increased grain yield) than those inoculated with a single AM isolate. Seed coating proved to be an appropriate tool to deliver AM fungi with benefits for chickpea plants at both experimental scales, but particularly relevant under field conditions. The mixture of multiple R. irregularis isolates was also used in consortium with Pseudomonas libanensis for coating cowpea seeds. This treatment significantly improved crop productivity in comparison with non-inoculated plants and plants inoculated with R. irregularis single-isolate + P. libanensis. The results showed improvements in grain lipid content, soil physicochemical properties (pH and soil organic matter), and crop yield under low-input agricultural systems. AM fungi and PGPR should be selected for microbial seed coating formulation according to their affinity with the host crop, growing conditions (e.g. soil properties) and farming practice (e.g. irrigation and fertilization), in order to obtain economical profits. This thesis is a contribution to the knowledge on microbial seed coating and highlights the potential of seed coating as a microbial delivery tool and the benefits of its use in different agricultural conditions. Microbial seed coating can be of great use for sustainable agricultural systems. Yet, in order to allow its large-scale application as a cost-effective technique for PGPR and AM fungi inoculation, further development is necessary.
O interesse em rizobactérias promotoras de crescimento de plantas (RPCP) e fungos arbusculares micorrízicos (FAM) para fins agrícolas (e.g. melhoramento do valor nutricional e rendimento de culturas, promoção de resiliência de plantas a factores abióticos) tem vindo a aumentar. Contudo, a aplicação em larga escala destes microrganismos tem sido dificultada pela escassez de dados sobre a performance destes em campo e viabilidade dos métodos de inoculação, em particular, no caso dos FAM. O revestimento de sementes, uma técnica que consiste em cobrir sementes com pequenas quantidades de materiais exógenos (incluindo inoculantes microbianos), representa uma potencial ferramenta para inocular microrganismos em grande escala. O revestimento de sementes tem vindo a ganhar importância no sector agrícola. Segundo a revisão da literatura realizada nesta tese, o revestimento de sementes foi usado em mais de 50 espécies de plantas (e.g. trigo, tomate, milho, melão, feijão, trevo), abrangendo sementes com diferentes características (dimensões, formas, texturas). Principalmente estudado para aplicação de várias espécies de RPCP (em particular do género Pseudomonas e Bacillus), o revestimento de sementes não tem sido tão frequentemente usado para inoculação dos FAM ou consórcios microbianos. O aumento da produtividade e proteção de culturas agrícolas contra agentes patogénicos têm sido o principal foco da investigação sobre revestimento de sementes com microrganismos benéficos. Por outro lado, os estudos referentes ao melhoramento da resistência de plantas a stresses abióticos através do revestimento de sementes têm sido consideravelmente menores. Um dos principais objectivos desta tese foi avaliar a técnica de revestimento de sementes como método de inoculação para FAM e RPCP. De uma forma geral, o revestimento de sementes permitiu a aplicação de pequenas quantidades de FAM e RPCP em sementes de três culturas: milho, feijão-frade e grão-de-bico. Uma comparação entre inoculação directa no solo (convencional) e revestimento de sementes com FAM, mostrou que, apesar da redução na quantidade de inoculo aplicado, a eficiência do fungo na colonização nas raízes da planta alvo foi similar. Contrariamente aos FAM, a presença de RPCP na rizosfera e raízes das culturas selecionadas não foi confirmada. É de conhecimento geral que tanto os FAM como RPCP têm a capacidade de melhorar a fertilidade do solo e o estado nutricional das plantas, o que pode ser de grande proveito para o desenvolvimento e crescimento destas. Através do aumento da disponibilidade ou eficiência de utilização dos nutrientes, estes microrganismos promotores de crescimento de plantas (MPCP) podem ajudar os agricultores a reduzir a dependência de fertilizantes sintéticos. De acordo com os resultados obtidos nesta tese, sementes revestidas com RPCP e FAM tiveram um impacto significativo no estado nutricional das plantas quando submetidas a diferentes regimes de fertilização. Por exemplo, sementes de milho revestidas com FAM (Rhizophagus irregularis) apresentaram um aumento significativo na concentração de nutrientes na parte área (azoto, fósforo, potássio, magnésio e zinco), quando comparado com plantas não inoculadas. A concentração de nutrientes, na parte área do milho, foi estimulada pela inoculação de R. irregularis, em particular, em tratamentos com fertilização reduzida ou ausente. Pelo contrário, o aumento na concentração de nutrientes em milho revestido com RPCP (Pseudomonas fluorescens) foi superior quando plena fertilização foi aplicada. Contudo, apesar do incremento nutricional, em geral, não se verificaram aumentos a nível da biomassa da planta em ambas as inoculações. Estes resultados confirmam que os inoculantes podem influenciar de forma positiva a absorção de nutrientes pelas plantas. RPCP e FAM são conhecidos por conferir resistência a plantas sobre stress hídrico. O revestimento de sementes com RPCP (Pseudomonas putida) mostrou, em geral, um efeito benéfico na productividade do feijão-frade, em especial, quando submetido a deficit hídrico moderado. Por sua vez, o revestimento de sementes com FAM (R. irregularis) individual ou em consórcio com P. putida promoveu a absorção de nutrientes pela planta, o conteúdo de pigmentos nas folhas e parâmetros de troca gasosa, contudo, na sua maioria na ausência de stress hidríco. Estes resultados realçam a importância de selecionar os MPCP que melhor fomentem a resiliência das plantas a stresses abióticos, a fim de tirar melhor partido da inoculação. Experiências de campo são indispensáveis para corroborar os benefícios do revestimento de sementes com microrganismos e a viabilidade para aplicações em grande escala. Uma comparação entre grão-de-bico revestido com um único isolado de R. irregularis e com uma mistura de vários isolados de R. irregularis, em estufa e em campo, mostrou que as plantas revestidas com múltiplos isolados tiveram um melhor desempenho (e.g. incremento na biomassa e na produção de grão). O revestimento de sementes mostrou ser uma ferramenta adequada para a inoculação de FAM com vantagens para a produção de grão-de-bico em ambas as escalas experimentais, em particular, para as condições de campo. A mesma mistura de isolados de R. irregularis foi usada em consórcio com Pseudomonas libanensis para revestir sementes de feijão-frade. Este tratamento aumentou significativamente a produtividade da cultura em comparação com plantas não inoculadas e plantas inoculadas com único tipo de isolados de R. irregularis + P. libanensis. Os resultados revelaram melhoramentos no conteúdo lipídico das sementes, nas propriedades físico-químicas do solo (pH e matéria orgânica do solo) e no rendimento da cultura, num sistema agrícola de baixo input. FAM e RPCP devem ser selecionadas de acordo com a sua afinidade com a planta alvo, condições de cultivo (e.g. propriedades do solo) e práticas agrícolas (e.g. irrigação, fertilização), de forma a obter lucros. Esta tese contribui para aumentar o conhecimento sobre revestimento de sementes com microrganismos, e realça o potencial da técnica como ferramenta de inoculação de FAM e RPCP e os seus benefícios em diferentes condições agrícolas. O revestimento de sementes com microrganismos pode ser de grande interesse para sistemas agrícolas sustentáveis. Contudo, de forma a permitir o uso em larga escala, como um método eficiente para a inoculação de RPCP e FAM, é necessário apostar no seu melhoramento e desenvolvimento.

O objectivo da primeira parte do trabalho consistiu em estabelecer condições de crescimento microaerofilo em fermentadores, para a produção industrial de Bradyrhizobium japonicum com vista à obtenção de células com citocromo oxidase cbb3 expresso. Após optimização, foi obtida uma taxa de crescimento média de 0.015 h-1, que serviu de referência para o processo de produção à escala industrial. Os resultados obtidos nesta primeira parte do trabalho, foram alcançados pela implementação de um conjunto de metodologias associadas a processos biotecnológicos determinantes para a segunda parte do trabalho. O objectivo da segunda parte do trabalho consistiu em optimizar condições de crescimento de estirpes autóctones de Rhizobium (123Ts2, 19Ms e 55Mp) com o fim de produzir inoculantes com potencial interesse na produção de leguminosas. Para a estirpe Rhizobium leguminosarum bv. trifolii 123Ts2, as maiores concentrações celulares foram obtidas em meio TYG, 28ºC, pH 6 e 50 mM de NaCl. Estes valores, serviram de referência para os ensaios realizados à escala piloto, onde os melhores resultados foram obtidos para um caudal de ar de 2000 ml/min a pH 6 constante. Para a estirpe Sinorhizobium meliloti 19Ms as maiores concentrações celulares registaram-se nos crescimentos efectuados em meio TYG a 28ºC, pH 6 e a 150 mM de NaCl. Já para a estirpe Sinorhizobium meliloti 55Mp concluiu-se que os níveis de temperatura e pH mais elevados estudados permitem taxas de crescimento mais altas, sendo as maiores concentrações celulares atingidas nos crescimentos a pH 6. Nos ensaios à escala piloto procedeu-se à optimização das condições de crescimento para a estirpe Rhizobium leguminosarum bv. trifolii 123Ts2 de modo a se obterem resultados conclusivos que fornecessem informação útil para a produção de inoculantes.
The purpose of the first part of this work regarded the establishment of microaerophilic growth conditions in fermenters, to obtain large yields of Bradyrhizobium japonicum cells overexpressing cbb3 cytochrome oxidase. After optimization, an average growth rate of 0.015 h-1 was obtained, which was used as a reference rate for the large-scale processes. The results acquired from the first part of the work were attained by employing methodologies related to biotechnological processes, which were crucial for the second part of the work. The purpose of the second part consisted of optimizing growth conditions of wild-type strains of Rhizobium, namely 123Ts2 Rhizobium leguminosarum bv. trifolii and 19Ms, and 55Mp Sinorhizobium meliloti, with the objective of obtaining inoculants with a potential interest for leguminous production. The largest cell densities of strain 123Ts2 were obtained in TYG medium, at 28ºC, pH 6.0, and 50 mM NaCl. These values were used as reference for the largescale tests, where the best results were obtained with an air-flow of 2000 ml/min at constant pH of 6.0. Regarding Sinorhizobium strain 19Ms, the highest cell densities were reached in TYG medium, at 28ºC, pH 6.0, and 150 mM NaCl. As for Sinorhizobium strain 55Mp, it was observed that higher temperatures and pH result in higher growth rates, the highest cell densities being attained at pH 6.0. Large-scale growth conditions for Rhizobium leguminosarum bv. trifolii 123Ts2 were optimized to obtain conclusive results in the production of inoculants.

This work was produced as original thesis for the degree of Doctor in AgriChains-From Fork to Farm at the University of Trás-osMontes and Alto Douro
A agricultura global deve duplicar a produção de alimentos até 2050 por forma a alimentar a crescente população mundial. Neste sentido, alimentos com um elevado valor nutritivo, como por exemplo as leguminosas (proteína, minerais, vitaminas e compostos bioativos) aparecem como uma resposta a esta necessidade. É, no entanto, essencial aumentar a sua produtividade. Ao mesmo tempo, é também necessário reduzir a aplicação de fertilizantes inorgânicos, devido ao elevado impacto negativo que estes têm para o ambiente. Para atingir estes objetivos, é essencial tirar proveito das múltiplas interações benéficas que ocorrem entre as plantas e os microrganismos. Os microrganismos benéficos presentes no solo, nomeadamente os rizóbios e os fungos micorrízicos arbusculares, em simbiose com plantas leguminosas, resultam numa simbiose tripartida e podem ser uma ferramenta biológica para melhorar a produção das culturas, através da fixação biológica de azoto e da absorção de fósforo do solo. Esta simbiose também aumenta a resistência das culturas à seca e às altas temperaturas, melhora a produtividade e a qualidade das culturas e a fertilidade do solo e diminui a incidência de ervas daninhas, doenças e pragas, sem os impactos negativos para o ambiente provocados pela aplicação de fertilizantes químicos. Neste sentido, os objetivos deste trabalho foram selecionar estirpes melhoradas de Rhizobium leguminosarum e Bradyrhizobium spp. para melhorar a fixação biológica de azoto e o desempenho das cultivares de fava e feijão-frade, e efetuar a caracterização fenotípica e genotípica dos simbiontes microbianos usando uma abordagem polifásica baseada em propriedades fenotípicas e na análise molecular. No presente trabalho, foi assim efetuada a identificação molecular dos rizóbios presentes em plantas de feijão-frade e fava recolhidas em diversas regiões de Portugal com diferentes condições climáticas e diferentes tipos de solo, utilizando uma abordagem de “Multilocus Sequence typing” (MLST) com 9 genes (“housekeeping” e simbióticos), a fim de obter informações ao nível da espécie e da simbiovar, uma vez que a amplificação da região 16SrDNA isoladamente não providenciou poder de resolução suficiente. Após a identificação molecular, foram realizados estudos in vitro para verificar a capacidade infectiva dos isolados (postulados de Koch) e para selecionar os melhores inóculos para cada cultura, os quais foram depois testados em condições de estufa, com o objetivo de avaliar os efeitos das inoculações simples e das co-inoculações com os microrganismos selecionados no crescimento, produtividade e conteúdo em proteína da respetiva leguminosa. Foi identificada uma elevada diversidade de rizóbios nos diferentes campos e regiões. Para o feijão-frade, foram selecionadas duas estirpes de rizóbios, Bradyrhizobium sp. e Bradyrhizobium elkanii. Para a faveira, foram selecionadas as bactérias Rhizobium laguerreae e Burkholderia sp.. Relativamente aos inóculos micorrízicos, uma mistura de Rhizophagus irregularis BEG140, Funneliformis geosporum BEG199 e Claroideoglomus claroideum BEG210 (1: 1: 1) foi desenvolvida e preparada pela Symbiom (Sázava, República Checa) para a cultura da faveira. Para o feijão-frade, o fungo micorrrízico (Claroideoglomus claroideum BEG210) foi cedido pelo Dr. Rui Oliveira, da Universidade de Coimbra, Portugal. No trabalho realizado em estufa com inoculação e co-inoculação com Rhizobium laguerreae e AMF, as plantas de faveira inoculadas com a bactéria mostraram um aumento significativo no número de folhas, área foliar, massa foliar por área e razão de área foliar, bem como em todos os parâmetros de produtividade avaliados. A inoculação simples dessas plantas com AMF também aumentou significativamente os parâmetros de produtividade. A co-inoculação mostrou melhorias significativas na proporção da área foliar e em todos os parâmetros de produtividade quando comparado com o controlo, mas não foi significativamente diferente das inoculações individuais. Nos estudos com feijão-frade, em condições de estufa, usando solo não esterilizado, a co-inoculação das plantas com Rhizobium sp. e AMF, Bradyrhizobium elkanii e AMF e Bradyrhizobium sp. e AMF aumentaram o teor de proteína das sementes em plantas sujeitas a déficite hídrico (25% da capacidade de campo) em 13, 17 e 30%, respetivamente. Considerando todas as análises realizadas neste trabalho em ambas as culturas, é possível concluir que a inoculação simples e combinada de plantas leguminosas com os microrganismos selecionados mostrou ter um grande potencial como ferramenta biológica para melhorar o crescimento e a produtividade das plantas leguminosas sujeitas a stress abiótico, mitigando os efeitos das alterações climáticas e reduzindo a necessidade de aplicação de fertilizantes de síntese.
Global agriculture has to double food production by 2050 in order to feed the world’s growing population. In this sense, food with a high nutritional value, such as the leguminous plants (protein, minerals, vitamins and bioactive compounds) appear as an answer to this need. However, it is crucial to increase its productivity. At the same time, it is also necessary to reduce the application of inorganic fertilizers, due to the high negative impact they have on the environment. To achieve these goals, it is essential to take advantages from the multiple beneficial interactions that occur between plants and microorganisms. Beneficial microorganisms present in the soil, namely rhizobia and arbuscular mycorrhizal fungi, in symbiosis with leguminous plants, results in a tripartite symbiosis and can be a biological tool to enhance crop production, through biological nitrogen fixation and phosphorus uptake from soil. This symbiosis also increases the resistance of crops to drought stress and high temperatures, improves crop productivity and quality and soil fertility and decreases the incidence of weeds, diseases and pests, without the negative impacts in the environment provoked by chemical fertilizer inputs. In this sense, the objectives of this work were to select improved strains of Rhizobium leguminosarum and Bradyrhizobium spp. for enhanced biological nitrogen fixation and field performance on cultivars of faba beans and cowpeas, and to perform the phenotypic and genotypic characterization of microbial symbionts using a polyphasic approach based on phenotypic properties and molecular analysis. In the present work, the molecular identification of rhizobial bacteria present in cowpea and faba bean plants collected from several regions of Portugal with different climatic conditions and different types of soil was performed using a Multilocus Sequence typing” (MLST) approach with 9 genes (housekeeping and symbiotic genes), to obtain information at species and symbiovars level, since the amplification of 16SrDNA region alone did not provide enough resolution power. After the molecular identification, in vitro studies were performed to check the ability of the isolates to nodulate other plants (Koch's postulates) and to select the best inoculants for each crop, which were after tested under greenhouse conditions, with the purpose of evaluating the effects of single and co-inoculation with the selected microorganisms on the growth, yield and protein content of the respective leguminous plants. High diversity of rhizobial bacteria was identified in different fields and regions. For cowpea plants, were selected two rhizobial strains, Bradyrhizobium sp. and Bradyrhizobium elkanii. For faba bean, were selected Rhizobium laguerreae and Burkholderia sp.. Regarding the mycorrhizal inoculants, a mix of Rhizophagus irregularis BEG140, Funneliformis geosporum BEG199 and Claroideoglomus claroideum BEG210 (1 : 1 : 1) was developed and prepared by Symbiom (Sázava, Czech Republic) for the faba bean crop. For cowpea, the mycorrhizal fungi (Claroideoglomus claroideum BEG210) was provided by Dr. Rui Oliveira, from de University of Coimbra, Portugal. In the greenhouse work developed with inoculation and co-inoculation with Rhizobium laguerreae and AMF, faba bean plants single inoculated with the bacteria showed a significant increase in the number of leaves, leaf area, leaf mass per area and leaf area ratio, as well as in all evaluated yield parameters. Single inoculation of these plants with AMF also significantly increased the yield parameters. Co-inoculation showed significant improvement in leaf area ratio and in all productivity parameters when compared with the control, but it was not significantly different from the individual inoculations. In the studies with cowpea, under greenhouse conditions, using non-sterilized soil, the co-inoculation of plants with Rhizobium sp. and AMF, Bradyrhizobium elkanii and AMF and Bradyrhizobium sp. and AMF increased the crude protein content of the seeds in plants under drought stress (25% of field capacity) in 13, 17 and 30%, respectively. Considering all analyses performed in this work in both crops, it is possible to conclude that single and combined inoculation of leguminous plants with selected microorganisms showed great potential as a biological tool to improve the growth and yield of leguminous plant under abiotic stress, mitigating the effects of climate change and reducing the need for chemical fertilizer inputs.

Associations between leguminous plants and symbiotic nitrogen-fixing bacteria (rhizobia) are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Rhizobia improve plant yield furnishing fixed nitrogen; therefore, they are highly used as inoculants, especially in sustainable agriculture. Though this association is species-specific, legume roots are exposed to heterogeneous rhizobial populations where different compatible strains are present and, could be infected by more than one strain. It is known that within the same rhizobial species different strains may have different competition capabilities, but detailed analyses able to predict the rhizobial competitive phenotype based exclusively on their genome are still lacking. In this thesis, we performed a bacterial genome-wide association analysis (GWAS) to define which genetic traits are responsible for an improved competitive phenotype in the model species Sinorhizobium meliloti. A panel of thirteen S. meliloti strains, whose genome is completely sequenced, has been selected and tested against three S. meliloti reference competitor strains (Rm1021, AK83 and BL225C) in a Medicago sativa nodule occupancy test. The measure of competition phenotypes previously obtained in the competition tests, in combination with strains genomic sequences of strains tested, were used to build-up a k-mer-based statistical models for each set of competition experiments. The obtained models were then applied to evaluate the accuracy in predicting the competition abilities of strains in the three competition patterns (vsRm1021, vsAK83 and vsBL225C). The competitive abilities of S. meliloti strains against two partners, BL225C and Rm1021, were well predict by the predictive models, as shown by the coefficient of determination R2 (equal to 0.96 and 0.84, respectively). Four strains showing the highest competition phenotypes (> 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) versus BL225C were used to identify k-mers associated with the competition phenotype. The most significantly associated k-mers (p <0.05) were mapped on the genomic sequences of the S. meliloti strains used. Most of the k-mers were located on the symbiosis-related megaplasmid pSymA and on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (i.e. glycerol, fatty acids) suggesting that competition abilities reside in multiple genetic determinants comprising several cellular components. The identification of the best rhizobial inoculants in two new effective breeding lines of pea (Pisum sativum) used in Lithuania (DS 3637-2 and DS 3795-3) was also investigated. Six rhizobial strains, isolated from pea plants, which could be used as potential inoculants, were phylogenetically identified and extensive phenotypically characterized by Phenotype Microarray. All the strains belonged to the Rhizobium leguminosarum group, and were subdivided into three groups related to Rhizobium anhuiense, Rhizobium leguminosarum bv. viciae and R. sophorae/R. laguerreae. Differences observed with Phenotype microarray were linked to different phylogeny of the strains. In terms of symbiotic efficiency, six strains showed different symbiotic performances depending on the breeding line used. In particular, Rhizobium anhuiense strain Z1 (the reference strain) and Rhizobium leguminosarum bv. viciae 14ZE were the best symbiotic inoculants with breeding lines DS 3637-2 and DS 3795-3, respectively.

Inoculation with diazotrophic bacteria is well documented as a means to enhance growth and increase yields of various crops, especially when used as an alternative or a supplement to the use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. Nitrogen is the most limiting nutrient for increasing crop productivity, and the use of chemical sources of N fertilizers is expensive, and may contribute to environmental pollution. Therefore, there is a need to identify diazotrophic inoculants as an alternative or supplement to N-fertilizers for sustainable agriculture. The search for effective diazotrophic bacterial strains for formulation as biofertilizers has been going on for over 40 years and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 195 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within the KwaZulu-Natal Province, Republic of South Africa. Ninety five of the isolates were selected for further screening because they were able to grow on N-free media using different carbon sources. Isolates that were very slow to grow on N-free media were discarded. Of these, 95 isolates were screened in vitro for growth promotion traits tests including tests for ammonia production and acetylene reduction. The best 20 isolates that were also able to reduce acetylene into ethylene were selected for growth-promotion trials on maize under greenhouse conditions. Of the 20 isolates, ten isolates enhanced (P = 0.001) growth of maize above the Un-inoculated Control. Molecular tests were conducted to identify the ten most promising isolates selected in the in vitro study. In the greenhouse study, these diazotrophic isolates were screened for their ability to enhance various growth parameters of maize (Zea mays L.), following various inoculation techniques (drenching, seed treatment, foliar spray and combination of these). Inoculations with the five best diazotrophic isolates by various methods of application increased dry weight and leaf chlorophyll content (P < 0.001, P = 0.001), respectively, compared to the Untreated Control. Although, all methods of application of diazotrophic inoculants used in this study resulted in increased dry weight and leaf chlorophyll content, combined methods of application (seed treatment + drenching) and sole application (seed treatment) were significantly more (P < 0.05) efficient. The best five most promising isolates were identified for growth promotion of maize under greenhouse conditions. They were also assessed for their effects on germination of wheat in vitro and were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of wheat (Triticum aestivum L.). These five isolates were also investigated for their potential to enhance growth and yields of maize and wheat crops in field trials, when combined with a low dose of nitrogenous fertilizer. These isolates were further studied for their contribution for enhancing plant growth through nitrogen fixation by predicting N content in leaves using a chlorophyll content meter (CCM-200) and correlated to extractable chlorophyll level at R2 = 0.96. In this study, relative to the Un-inoculated Control, the best five isolates enhanced growth of maize and wheat when combined with a 33% N-fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry weight of maize and wheat; and enhanced root and shoot development of these crops in both greenhouse and field conditions. The best contributions of diazotrophic bacteria was achieved by Isolate LB5 + 0% NPK (41%), V9 + 65% NPK (28.9%), Isolate L1 + 50% NPK (25%), Isolate L1 + 25%NPK (22%) and LB5 + 75% NPK (15%) undergreenhouse conditions. At 30 or 60 DAP, isolates with 33%N-fertilizer caused relatively higher dry weight than the 100%NPK. Inoculation of Isolate StB5 without 33N% fertilizer cuased significant (P<0.005) increases in stover dry weight. In field studies, inoculation of diazotrophic bacteria alone or with 33%N-fertilizer resulted in relatively greater increases of dry weight, stover dry weight, number of spikes and yield at different growth stages higher than the Un-inoculated or Unfertilized Control. However, the increases were not statistically significant. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising yields. The isolates obtained in this study can effectively fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture.
Ph.D. University of KwaZulu-Natal, Pietermaritzburg 2013.