PROGRESSIVE REPORT
Type of report: final report on investigation project
Period covered: 17. 06. 1998. – 17. 06. 2001.
Title of project: “Effectiveness of inoculation in alfalfa breeding in ecological
conditions of the Bjelovar and Bilogora county”.
Contract number: 525-02-98-10
Project starts date: June 17th 1998.
Principal Investigator: Doc.dr. Sanja Sikora
Participating Institution: Faculty of Agriculture University of Zagreb,
Department of microbiology
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(date)
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(Signature of the Principal Investigator)
Investigation theme
Main goal of these investigations was to enhance alfalfa production in the area of
Bjelovar and Bilogora county. Area of Bjelovar and Bilogora county is characteristic
for cattle breeding and therefore production of effective and qualitative forage is of a
great importance for family farms. Altough there is a interest for alfalfa breeding, this
important and qualitative forage cultivar is not widely present. We have tried to
eliminate some limiting factors in alfalfa breeding by high soil acidity removal and by
introduction of alfalfa seed inoculation with efficient nodulating strains
(Sinorhizobium meliloti) in order to enable qualitative and cost effective production of
this forage on the family farms of the region.
Laboratory investigations followed by greenhouse experiment were carried out at the
Faculty of Agriculture University of Zagreb. Further investigations were carried out
through micro and macro field trials established on three family farms in the Bjelovar
and Bilogora county ( locations: Staro Štefanje, Veliki Grđevac and Višnjevac).
Technology description
In order of higher exploatation of symbiotic nitrogen fixation natural process it is
necessary to select the most efficiant S. meliloti strains for production of alfalfa seed
inoculant. Investigations on this project have started by compatibility and symbiotic
efficiency tests of different introduced S. meliloti strains trough greenhouse
experiment. Soil for this experiment was brought in from the area of Bjelovar and
Bilogora county in order to achieve results that could give directions for further
investigations on certain locality. Different chemicals for acidity removal were
included in the experiment as well.
Following the positive responses achieved in the greenhouse experiment, field micro
trial was established at the family farm Mirović (locality Višnjevac). In the case of
alfalfa seed inoculation it is important to pay attention to mineral nitrogen
fertilization. In order to ensure the cost effective production it is necessary to enhance
the portion of fixed and lower the portion of mineral nitrogen. It is well known that
high doses of mineral nitrogen, besides some other negative effects, can significantly
reduce atmospheric nitrogen fixation. According to that, inoculation requires low
mineral fertilization. Due to that fact, fertilization of the field micro trial was 570
kg/ha NPK 7:20:30. Trial factors were different liming chemicals and different S.
meliloti strains. Green mass yield, dry matter content as well as total nitrogen content
in aboveground part of the plant were observed during the trial. The best results were
achieved in application of calcium carbonate (10t/ha) and S. meliloti strain 2011.
Based upon microtrial results, the most effective combinations were chosen and
included in further testing through field macro trials. Identical trials were established
at the family farms in Staro Štefanje and Veliki Grđevac. Liming and basic
fertilization were carried out in autumn. Besides the high acidity regulation and basic
fertilization determination it is essential to pay attention to tillage and crop rotation in
order to achieve the best conditions for alfalfa production. Alfalfa seeding carried out
in spring (variety OS-88) followed inoculation with S. meliloti strain 2011.
Inoculation procedure is fast and simple according to directions given by producer. If
neccessary, trial was treated with herbicides during the vegetation period.
Economic analysis (cost effectiveness)
Suggested solution includes existing technology enhacement by introduction of
alfalfa seed inoculation as a regular agrotechnical measure. It is known that alfalfa as
one of the most important protein producers per hectare has a large nitrogen uptake
from soil. According to different authors, those values are estimated to 300 kg/ha for
the straw yield of 10 t/ha. Due to that, usual alfalfa fertilization includes multiple
application of significant quantities of mineral nitrogen fertilizer as well as cattle
manure which is emphasized on soils of lower quality. Alfalfa needs for nitrogen
could be satisfied through symbiotic nitrogen fixation by introduction of seed
inoculation. Regarding the symbiotic relationship between alfalfa and Sinorhizobium
meliloti, natural sources of energy and plant photosynthetic capacity are used for cost
free atmospheric nitrogen fixation. In contrast, 80 MJ of energy are needed for
production of 1 kg of mineral nitrogen. Besides, this kind of production requires
expensive (world market depending) fossile fuels. It is estimated that symbiotic
relationship of alfalfa and effective S. meliloti strain can yield 200-250 kg N/ha. If we
translate the ammount of pure nitrogen into expensive mineral fertilizer required for
succesfull alfalfa breeding than savings that could be achieved at the family farms
through inoculant applications become more than obvious. Liming is also required for
alfalfa breeding on acid soils. Although this measure requires expenses, its results
have long term and multiple effects. Besides turning unfavorable soil pH into
favorable, there is a calcium uptake that is lost trough plant in a large quantity (app.
300 kg /ha).
Application
Proposed solutions for alfalfa breeding enhacement in conditions of Bjelovar and
bilogora county enable unfavorable soil pH removal as well as cost effective
production by using natural process of symbiotic nitrogen fixation. Agrotechnical
measures used within the frame of these investigations could be succesfully applied at
family farms due to the available preparates and their application does not require
special equipment or skills.
Farmers evaluation
Family farms included in this project are satisfied with proposed solutions and
established cooperation. Important value of this cooperation is reflected in the fact
that some family farms have already started with application of agrotechnical
measures used in this project. Knowledge and data transfer are enabled through
established contacts between farmers and scientists involved in this project.
Quantitative data
During the first year of investigations, alfalfa in vegetation trial was harvested four
times. The highest green mass and straw yields are determined in second harvest. In
all harvests, the lowest values were determined for non inoculated, control variants.
Significant differences were not determined between different S. meliloti strains.
Nevertheless, it could be noted that in most cases, inoculation with D-528 strain
resulted with the highest yield of green mass and straw. In the first harvest, straw
yield differences between variants inoculated with D-528 and 2011 strains compared
to variants inoculated with D-469 strain were statistically significant. Regarding
liming, first harvest showed significant influence of “Agrarvital” on yield increase
while other harvests showed higher yields with calcium carbonate application. There
was a statistically significant interaction of inoculation and liming in the fourth
harvest. The highest yields were determined on variants where S. meliloti strain D-528
and calcium carbonate were used. Total nitrogen content in aboveground part of the
plant was determined in second and fourth harvest. Values obtained in second harvest
were from 1,96% to 2,6% while significantly higher total nitrogen content was
determined in the fourth harvest (2,60 – 3,77%). During the second year of
investigations there was five alfalfa harvests and all data were statistically processed.
Significant differences between certain harvests were determined for the green mass
yield due to the extremely high temperatures during the vegetation period. The
highest green mass yields were determined in the first harvest while following
harvests were significantly reduced due to the unfavorable climatic conditions. The
highest values for green mass and straw yields were determined for S. meliloti strain
2011. Regarding the influence of liming, significantly highest yields were achieved in
the first harvest by the application of calcium carbonate while other liming chemicals
showed no significant differences in other harvests. Total nitrogen content fluctuated
concerning different combinations from 1,94 to 2,82% and the lowest values were
determined on control variants. In the third year of investigations, inoculation
influence was lower than years before although application of the 2011 S. meliloti
strain resulted in highest yields. The best results regarding liming chemicals were
obtained on variants with calcium carbonate.
Field microtrial on family farm Mirović (locality Višnjevac) was established in
autumn of 1999. During the year 2000 alfalfa was harvested four times. In the first
harvest, statistically processed data for green mass and dry matter yields showed
significant influence of inoculation and liming as well as for their interaction.
Significantly highest yields were determined for application of strain 2011 and
calcium carbonate. Mentioned combination resulted with the highest total nitrogen
content (3,25%). Same results were obtained in other three harvests which undoubtely
shows that the most effective combination for Višnjevac locality is inoculation with
2011 strain and application of calcium carbonate. First harvest of the next year
(2001.) had highly significant differences in green mass and dry matter yield obtained
by application of the same factors as well. In the other harvests during the 2001.
lowest green mass and dry matter yields were determined on control variants while
there were no statistically significant differences between strains. Application of
calcium carbonate resulted in highest values for green mass yield as well as for dry
matter yield.
In the spring of 2000., macrotrials were established on localities Staro Štefanje and
Veliki Grđevac. Through this macrotrials on larger areas, in investigatons were
included those factors that gave the best results in microtrials. During the first year of
investigations there was a statistically significant influence of inoculation with strain
2011 and liming on the green mass yield at the Staro Štefanje locality. Inoculation
resulted in significantly higher dry matter yield. During the 2001., higher yields were
determined on variants with inoculation and liming but those differences were not
statistically significant. Interaction of both factors was statistically significant. At the
Veliki Grđevac locality, during the 2000, statistically significant influence of
inoculation on green mass yield was determined in all three harvests. Inoculation had
a positive influence on dry matter yield as well in all harvests while differences in
second and third harvest were statistically significant. Liming caused significant yield
increase in green mass as well as in dry matter in first two harvests while in the third
harvest yield increase was not statistically significant. During the 2001., inoculation
caused yield increase but significant differences were determined in second harvest.
Calcium carbonate application resulted in significant yield increase in first two
harvests while differences in third harvest were not statistically significant.
Qualitative data
Identical field macrotrials were established on locations Staro Štefanje and Veliki
Grđevac. Alfalfa breeding was very succesfull in Veliki Grđevac while trial in Staro
Štefanje did not give expected response. Reason for that was disabled proper tillage.
This trial was established on a extremely heavy pseudogley characterized by
unpermeabile layer, large clay portion and therefore low air capacity. In those
conditions, extremely deep tillage is required which could not be done due to the lack
of adequate mechanisation. In order to assure enough forage cultivars on those areas,
extension of trialed table was sown with mixture of annual rye grass and white clover.
This grass and clover mixture is not so demanding as alfalfa and could be
recommended for such extremely unfavorable areas.
Alfalfa was succesfuly bred on family farms in Veliki Grđevac and Višnjevac. Field
microtrial in Višnjevac showed clear differences between some trial variants even
before all data was collected. In complete investigation period, noninoculated variants
were significantly lower developed with yellow leaf coloring while inoculated
varinats were well developed with dark leaf coloring.
Selection of proper S. meliloti strain is of a great importance for succesfull
inoculation. Strains acquired from different european countries were used within this
project. During this investigations, isolation of indigenous strains and formation of
own culture collection have started in order to use effective as well as well adapted
strains in the future.
Conclusions
Results of this investigations point to possible alfalfa breeding enhacement in area of
Bjelovar and Bilogora county with application of liming and seed inoculation with
symbiotic nitrogen fixators together with other agrotechnical measures which have to
be adapted to certain conditions on the site. Results of this investigations confirm that
liming is required for succesfull alfalfa breeding. Application of inoculation requires
further investigations in order to select the most appropriate strains for certain area as
well as to enable observation of inoculation influence through time period. Regardind
alfalfa as perennial crop, time frame of this investigations represents certain limiting
factor. In order to achieve more precise results, two year period in such unfavorable
conditions as they are in the region of Bjelovar and Bilogora county is not enough and
should be continued.