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STEM NODULATING BACTERIAAND THEIR
APPLICATION IN AGRICULTURE
RAVIKUMARHOOGAR
PALB 5191
PRESENTATION ON
Introduction
Infection Process And Nodule Formation.
Factors Affecting Symbiosis
APPLICATIONIN AGRICULTURE
Inoculation
FLOW OF PRESENTATION
• The symbiosis between leguminous plants and
soil bacteria of the Rhizobiaceae leads to the
formation of nitrogen-fixing nodules, generally
exclusively appearing on the roots.
• A few legume species, however, form nodules
not only on their roots, but also at stem-located
root primordia.
• The first example of this phenomenon was first
reported in 1928 in Aeschynomene aspera L.
by Hagerup.
INTRODUCTION
• Only a few legume species bear nodules both on
their roots and stems. They belong to the three
genera Sesbania (one species), Aeschynomene
(about 15 species) and & Neptunia (one species).
• These plants have in common the ability to grow in
waterlogged soils and are potential candidates for
green manuring in paddy fields.
• Up to now, 26 different stem-nodulating
legume species have been reported belonging
exclusively to the genera Aeschynomene,
Neptunia, and Sesbania.
The most distinctive characteristic of stem-nodulating
legumes is the presence of predetermined nodulation
sites on the stems.
The formation of these sites is independent of infection
with rhizobia They comprise primordia of adventive roots
that are able to grow out under waterlogged conditions .
 These primordia can be distributed over the whole
length of the stem or appear only on lower stem portions.
They are arranged in straight vertical rows (Sesbania) or
in spiral-shaped rows winding around the stem
(Aeschynomene), giving the appearance of random
distribution
Fig 1. Nodulated stems of 50-day-old Sesbania rostrata (A)
and Aeschynomene afraspera (B).
Table1:Stem-nodulating legumes, their geographic
distribution.
Azorhizobium caulinodans
The fast growing Rhizobium strain ORS571,now called
Azorhizobium caulinodans, was isolated from stem nodules
of the tropical legume Sesbania rostrata. The strain can grow
in the free-living state at the expense of molecular nitrogen.
The nitrogenase, purified from cells grown in a fermenter,
was shown to be composed of two components, a MoFe-
protein and a Fe-protein.
The enzyme activity was found to be subject to"switch-off"
when ammonia was added to a N2-fixing culture.
Electron micrograph of negatively stained Azorhizobium caulinodans ORS
571T grown in liquid medium.
For stem inoculation, shoots can be sprayed with
a suspension containing about 108 bacteria/ml,
using either a liquid culture of rhizobia, a
colloidal suspension obtained by mixing
entrapped rhizobia in a phosphate buffer (0.06m,
pH 6.8), or a suspension of crushed stem nodules
with water passed through a filter.
Inoculation
Fig 2. Typical appearances of stem nodules formed by mutants
categorized by type.
Table:2 Effects of A. caulinodans inoculation on N, P and K
accumulation of S. rostrata on 60 DAS
Tukey–Kramer HSD test (P<0.05).
Infection Process, Nodule Formation, and Fine Structure of Stem
Nodules
• Stem-nodulating rhizobia are true soil bacteria;
without the appropriate macrosymbiont they
thrive saprophytically on soil organic matter.
• Due to their unipolar flagellum they are highly
mobile in the aquatic phase.
• Besides the soil, the phyllosphere of the host
plant seems to be an alternate ecological niche
for nonsymbiotic growth of stem-nodule
rhizobia.
• Inoculum for stem infection can come from two
sources: the soil and the phyllosphere. In both
cases, rain seems to play major role for stem
inoculation.
• soil splash due to rain may bring rhizobia to the
lower stem.
• ants as a possible vector to inoculate primordia
on the stem of S. rostrata under nonflooded
conditions.
• Wind may be another important factor; often a
high nodulation rate can be observed on stems of
stem nodulating legumes along dusty roads.
• Wind may transport contaminated soil particles
on above-ground plant parts thus favoring stem
inoculation.
• After the rhizobia have reached the nodulation
site, they penetrate into the cortical tissues of
the stem.
• Upon penetration of rhizobia in the intercellular
space, infection pockets are formed into the
basal layers of the root primordium, which then
resume their meristematic activity. As a result,
cell division starts in the infective center and the
nodule begins to form.
• intracellular infection threads are formed.
• At the infective stage, rhizobia change in shape
and size. They become bacteroids and are
surrounded by a membrane envelope.
• As the growing infective center forms the
nodule, the majority of the cells in stem
nodules are infected.
• The formation of stem nodules becomes
macroscopically visible within 5 to 7 days
after inoculation and the nodules reach their
full size in 15 to 20 days.
• In S. rostrata, stem nodules are spherical
protrusions, 5 to 12 mm in diameter. They can
easily be detached from the stem, as the basal
portion of the nodule forms a narrow neck.
• In Aeschynomene flattened hemispheric nodules
are formed. Because no neck formation is found,
the nodules are hard to remove.
Factors Affecting Symbiosis
 Survival of Rhizobia : Like root-nodule
bacteria, stem-nodulating bacteria are true soil
bacteria. A carbon source is critical for their
survival in the soil.
Phyllospheric or Epiphytic Survival :
contain nutritional substances like amino acids
and carbohydrates.
Mineral Nitrogen :
• Normally, crop plants respond to fertilizer
nitrogen in the field since most of the cultivated
soils worldwide are deficient in nitrogen.
• For reasons of energy legumes prefer soil or
fertilizer nitrogen rather than biologically fixed
nitrogen.
• Mineral N can reduce both nodulation and N2
fixation in stem nodulating legumes; the degree
of inhibition varies with the compound and its
concentration.
APPLICATIONIN AGRICULTURE
Agronomic Use of the Stem-Nodulating
Legumes
Leguminous GM crops are potential N sources
with relatively high efficiency, but also have
diverse non-N effects such as the improvement of
soil properties and control of weeds, pests, and
diseases. Positive effects on soil properties
include,
maintenance or improvement of soil organic
matter.
conservation and recycling of nutrients.
increased mineralization.
increased biological activity.
increased cation exchange capacity.
reduction in soil erosion, and reduction in
fertilizer N losses.
Potential Rice Environments for GM Use
• The irrigated lowland environment, characterized by
an assured water supply, has a high cropping
intensity.
• The main problem of this environment is crop
competition for time, space, and labour. In areas
where the gap is very short or where early-season
waterlogging occurs, fast-growing Sesbania or
Aeschynomene would fit between the main crops. S.
rostrata may be used as GM before wet season rice,
during the long-day period (April/May) and A.
afraspera before the dry season rice
(November/December) during the short-day period.
Growing GM as an Intercrop and on
Bunds/Wastelands.
Optimum Time for GM Incorporation and
Rice Transplanting.
Sesbania green manure is added to field
Table 3:Onion yield under different treatments
Stem nodulating

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Stem nodulating

  • 2. STEM NODULATING BACTERIAAND THEIR APPLICATION IN AGRICULTURE RAVIKUMARHOOGAR PALB 5191 PRESENTATION ON
  • 3. Introduction Infection Process And Nodule Formation. Factors Affecting Symbiosis APPLICATIONIN AGRICULTURE Inoculation FLOW OF PRESENTATION
  • 4. • The symbiosis between leguminous plants and soil bacteria of the Rhizobiaceae leads to the formation of nitrogen-fixing nodules, generally exclusively appearing on the roots. • A few legume species, however, form nodules not only on their roots, but also at stem-located root primordia. • The first example of this phenomenon was first reported in 1928 in Aeschynomene aspera L. by Hagerup. INTRODUCTION
  • 5. • Only a few legume species bear nodules both on their roots and stems. They belong to the three genera Sesbania (one species), Aeschynomene (about 15 species) and & Neptunia (one species). • These plants have in common the ability to grow in waterlogged soils and are potential candidates for green manuring in paddy fields.
  • 6. • Up to now, 26 different stem-nodulating legume species have been reported belonging exclusively to the genera Aeschynomene, Neptunia, and Sesbania.
  • 7. The most distinctive characteristic of stem-nodulating legumes is the presence of predetermined nodulation sites on the stems. The formation of these sites is independent of infection with rhizobia They comprise primordia of adventive roots that are able to grow out under waterlogged conditions .  These primordia can be distributed over the whole length of the stem or appear only on lower stem portions. They are arranged in straight vertical rows (Sesbania) or in spiral-shaped rows winding around the stem (Aeschynomene), giving the appearance of random distribution
  • 8. Fig 1. Nodulated stems of 50-day-old Sesbania rostrata (A) and Aeschynomene afraspera (B).
  • 9. Table1:Stem-nodulating legumes, their geographic distribution.
  • 10. Azorhizobium caulinodans The fast growing Rhizobium strain ORS571,now called Azorhizobium caulinodans, was isolated from stem nodules of the tropical legume Sesbania rostrata. The strain can grow in the free-living state at the expense of molecular nitrogen. The nitrogenase, purified from cells grown in a fermenter, was shown to be composed of two components, a MoFe- protein and a Fe-protein. The enzyme activity was found to be subject to"switch-off" when ammonia was added to a N2-fixing culture.
  • 11. Electron micrograph of negatively stained Azorhizobium caulinodans ORS 571T grown in liquid medium.
  • 12. For stem inoculation, shoots can be sprayed with a suspension containing about 108 bacteria/ml, using either a liquid culture of rhizobia, a colloidal suspension obtained by mixing entrapped rhizobia in a phosphate buffer (0.06m, pH 6.8), or a suspension of crushed stem nodules with water passed through a filter. Inoculation
  • 13. Fig 2. Typical appearances of stem nodules formed by mutants categorized by type.
  • 14. Table:2 Effects of A. caulinodans inoculation on N, P and K accumulation of S. rostrata on 60 DAS Tukey–Kramer HSD test (P<0.05).
  • 15. Infection Process, Nodule Formation, and Fine Structure of Stem Nodules • Stem-nodulating rhizobia are true soil bacteria; without the appropriate macrosymbiont they thrive saprophytically on soil organic matter. • Due to their unipolar flagellum they are highly mobile in the aquatic phase. • Besides the soil, the phyllosphere of the host plant seems to be an alternate ecological niche for nonsymbiotic growth of stem-nodule rhizobia.
  • 16. • Inoculum for stem infection can come from two sources: the soil and the phyllosphere. In both cases, rain seems to play major role for stem inoculation. • soil splash due to rain may bring rhizobia to the lower stem. • ants as a possible vector to inoculate primordia on the stem of S. rostrata under nonflooded conditions. • Wind may be another important factor; often a high nodulation rate can be observed on stems of stem nodulating legumes along dusty roads.
  • 17. • Wind may transport contaminated soil particles on above-ground plant parts thus favoring stem inoculation. • After the rhizobia have reached the nodulation site, they penetrate into the cortical tissues of the stem.
  • 18. • Upon penetration of rhizobia in the intercellular space, infection pockets are formed into the basal layers of the root primordium, which then resume their meristematic activity. As a result, cell division starts in the infective center and the nodule begins to form. • intracellular infection threads are formed. • At the infective stage, rhizobia change in shape and size. They become bacteroids and are surrounded by a membrane envelope.
  • 19. • As the growing infective center forms the nodule, the majority of the cells in stem nodules are infected. • The formation of stem nodules becomes macroscopically visible within 5 to 7 days after inoculation and the nodules reach their full size in 15 to 20 days. • In S. rostrata, stem nodules are spherical protrusions, 5 to 12 mm in diameter. They can easily be detached from the stem, as the basal portion of the nodule forms a narrow neck.
  • 20. • In Aeschynomene flattened hemispheric nodules are formed. Because no neck formation is found, the nodules are hard to remove.
  • 21. Factors Affecting Symbiosis  Survival of Rhizobia : Like root-nodule bacteria, stem-nodulating bacteria are true soil bacteria. A carbon source is critical for their survival in the soil. Phyllospheric or Epiphytic Survival : contain nutritional substances like amino acids and carbohydrates.
  • 22. Mineral Nitrogen : • Normally, crop plants respond to fertilizer nitrogen in the field since most of the cultivated soils worldwide are deficient in nitrogen. • For reasons of energy legumes prefer soil or fertilizer nitrogen rather than biologically fixed nitrogen. • Mineral N can reduce both nodulation and N2 fixation in stem nodulating legumes; the degree of inhibition varies with the compound and its concentration.
  • 24. Agronomic Use of the Stem-Nodulating Legumes Leguminous GM crops are potential N sources with relatively high efficiency, but also have diverse non-N effects such as the improvement of soil properties and control of weeds, pests, and diseases. Positive effects on soil properties include, maintenance or improvement of soil organic matter. conservation and recycling of nutrients.
  • 25. increased mineralization. increased biological activity. increased cation exchange capacity. reduction in soil erosion, and reduction in fertilizer N losses.
  • 26. Potential Rice Environments for GM Use • The irrigated lowland environment, characterized by an assured water supply, has a high cropping intensity. • The main problem of this environment is crop competition for time, space, and labour. In areas where the gap is very short or where early-season waterlogging occurs, fast-growing Sesbania or Aeschynomene would fit between the main crops. S. rostrata may be used as GM before wet season rice, during the long-day period (April/May) and A. afraspera before the dry season rice (November/December) during the short-day period.
  • 27. Growing GM as an Intercrop and on Bunds/Wastelands. Optimum Time for GM Incorporation and Rice Transplanting.
  • 28. Sesbania green manure is added to field
  • 29. Table 3:Onion yield under different treatments