Drought and waterlogging are major abiotic stresses that limit the productivity of Brachiaria forage grasses. Little attention has been given to separate productivity under drought or waterlogging, from coping mechanisms in Brachiaria forage grasses. Wide phenotypic variation exists among Brachiaria grasses to cope with these stresses. This presentation will cover : 1) the current knowledge of morpho-physiological mechanisms and functional adaptations of Brachiaria spp cultivars to cope with these stresses and 2) the use of sensors and digital image analysis for the non-destructive and automated analysis of Brachiaria growth and performance at different time scales.

1. J. A. Cardoso, J. C. Jiménez, K. Odokonyero,
L.M. Pineda, Hannah Vos, Fernando Vergara,
Daniela Chamorro, I. M. Rao
Collaborators:
CIAT: J. Polanía, J. Arango, J. Nuñez, Plant
nutrition lab, Nutrition quality lab, Birthe Paul
Beca-ILRI Hub
KARI-Kenya
RAB-Rwanda
AgResearch-New Zealand
Corpoica-Colombia
INTA-Nicaragua
IDIAP-Panamá
Mechanisms of adaptation to drought
and waterlogging in Brachiaria grasses

2. Brachiaria spp.:
Important forage grasses in the tropics
• African origin
• Estimated 100 million hectares in
Brazil alone.
• Breeding program started in the
late 1980s.
• Wide range of adaptation to
climatic and edaphic factors.
• Carbon accumulation in soil,
reduction of greenhouse effect
gases from soil (N20) and
methane from livestock

3. Why do we need to know mechanisms
of abiotic stress adaptation?
• Identifying plant attributes
that contribute to
resistance/tolerance to
major abiotic stresses (e.g.,
drought and waterlogging)
• Developing rapid, reliable
and high throughput
screening methods
Phenotyping of Brachiaria
genotypes developed by the
breeding program

4. Drought resistance
(avoidance/tolerance)
Assessment methods
• Leaf gas exchange/porometry
• Infrared thermometry
• Carbon isotope
discrimination???
• Chlorophyll content (SPAD)
• Chlorophyll fluorescence
• Relative water content in leaves
• Weighing each container on a
regular basis
• Vertical distribution of roots in
soil cylinders (120 cm height x
22 cm width; 80 cm height x 7.5
cm width)
• Micrographs from root cross
sections
• High stomatal conductance
• Delayed leaf senescence
• High quantum yield
• High osmotic adjustment
• High transpiration efficiency
• Deep root systems
• Increased root length density in
medium and deep soil layers
• Decreased resistance to water
movement from soil by increasing
root hair growth and xylem
diameters
Brachiaria hybrid
cv. Cayman
Drought

5. Shoot growth and biomass partitioning after 5 weeks of drought
Experiment 1
35 kg of soil
Experiment 2
62 kg of soil

6. Based on hue,
chlorophyll contents
(SPAD or analytical) can
be estimated (r2 > 0.85)
Binary images are used
to estimated shoot areas
(difference of dark and
white pixels) (r2 > 0.7)
Skeletonize to determine
leaf apparition rates
Non-destructive assessment of shoot growth
over time (semi-automated)
scripts at Github, juan_cardosinho
1
2
3
4
5
0 days 7 days 14 days 21 days

7. Projectedshootarea(cm2)
0 days 10 days 21 days
Assessment of shoot growth under
progressive drought
• Faster growth rates for Napier grass, Rhodes grass and hybrid Cayman
• Greater shoot area represents greater demands for water
Wilting plants

8. Root growth after 5 weeks of growth

9. Root growth after 5 weeks of growth

10. Root growth after 5 weeks of growth

11. Estimation of root length using digital images

12. Photosyntheticrate(µmolCO2m2s-1)
Leaf gas exchange and chlorophyll fluorescence
Transpirationrate(mmolH2Om2s-1)
Photosyntheticefficiency
• Preliminary results indicate that reduction of
growth under drought is mainly due to
restriction of leaf gas exchange
• Photosynthetic efficiency (Fv/Fm) is relatively
insensitive to drought stress indicating
stomatal limitation of photosynthesis
0.
0.
0.
0.
0.
0.
0.
0.
0.
Brachiaria grasses

13. 32.7º C
Growth of Napier grass under drought conditions for 21 daysGrowth of Mulato II under drought conditions for 21 days
Wilting
plant
Growing
plant
Mulato II shows superior resistance to terminal drought
conditions than Napier grass
• Regulated use of water allows
Mulato II to sustain growth under
drought conditions.
• Mulato II responds to drought by
early closing of stomata which
reduces transpiration.
• Leaf and canopy temperature
values are measured using
infrared thermography.
Thermal infrared images

14. CTDdrought
Napier
Rhodes
Cayman
Marandu
Toledo
Llanero
Mulato II
Basilisk
Piata
Mulato
Tully
Napier
Rhodes
Cayman
Marandu
Toledo
Mulato II
Piata
Mulato
Tully
Tupi
Llanero
Basilisk
Canopy and leaf temperature as proxies
for deep rooting
• Canopy temperature depression (CTD) = Air temperature - Canopy temperature
• A cooler canopy (higher CTD) indicates transpiring leaf and indicates access to
water by roots
Observable root length
(m plant-1)
Root depth
(m plant-1)
Observable root length
80-120 cm root depth
(m plant-1)

15. CaymanMulato II
MulatoII
Time course of growth and water uptake under drought
conditions

16. Water uptake and growth under
drought conditions
• Currently, a simultaneous analysis of shoot and root growth and water content
across the soil profile is possible using digital image analysis and TDR.
7 days 14 days
Mulato II Napier Mulato II Napier

17. Root traits that influence water transport
XV
• More and greater xylem vessels
(XV) conduct more water
(decreased axial resistance)
Increased xylem vessel (XV) under
drought conditions for Napier grass
• Increased root diameter facilitates
penetration in drying soils
Greater root diameter in Napier
grass
• Roots hairs for decreasing
radial resistance
Longer and denser root hairs in
Mulato II
Irrigated Drought
NapiergrassMulatoII

18. MulatoIINapiergrass Irrigated Drought (10 days)
0 h 1 h 2 h 3 h
1cm
Root elongation rate
• Faster root elongation rates for Napier
• Higher inhibition of root growth under drought conditions for Mulato
II (65%) than Napier (35%)

19. Irrigated Drought
Irrigated Drought
NapiergrassMulatoIIB.humidicola Aerenchyma development
AER
• Aerenchyma (AER) can improve
the acquisition of water and
nutrients by reducing the
metabolic costs of soil
exploration.
AER
t

20. % Inhibition root elongation
0
5
10
15
20
25
0 50 100
%Aerrootsdrought
Napier B. humidicola
Mulato II
• Aerenchyma (AER) can improve the acquisition of water and nutrients by
reducing the metabolic costs of soil exploration.
• We plan to quantify respiration rates of aerenchymatyous roots vs. non
aerenchmatous roots using an IRGA.
Aerenchyma development and root growth

21. Root angles
MulatoIINapiergrass Irrigated Drought (10 days)
• Phenotypic plasticity for root angles
• Straighter angles under drought conditions
• Evaluation for root angles under irrigated and drought conditions underway
1m

22. Recovery after drought
• Short term drought restricts growth of Mulato II and is reflected in final biomass

23. Mechanisms Drought resistance in Brachiaria
Water spenders
Maintaining water uptake
Napier grass
Cayman
Water savers
Reducing water loss
• Piatá
• Deep roots
• Increased root length
density at depth
• Increased root growth at
expense of shoots
• Closing of stomata
• Leaf senescence
• Reduced leaf area
• Cayman
Both mechanisms
• Napier grass
• Rhodes grass
Terminal drought Intermittent drought
• Mulato II
• Basilisk
• Mulato
• Marandu
• Tupi
• Toledo
• Tully
• Llanero
Cultivars
Productivity

24. Effects of waterlogging on Brachiaria grassesTullyToledoRuzigrass

25. Drained (D) Waterlogged (W) D W D W
Tully Toledo Ruzi grass
Tolerant Mod. tolerant Sensitive
• Increased root death and smaller root system in non-tolerant genotypes
• Reduction in shoot growth in non-tolerant genotypes
• Increased leaf senescence, chlorophyll loss, stomatal closure, lower values
of photosynthetic efficiency (fv’/fm’) in non-tolerant genotypes.
21 days of treatment
Effects of waterlogging on Brachiaria grasses

26. Traits associated with waterlogging
tolerance in Brachiaria grasses
• Aerenchyma (air spaces) allows oxygen difussion
from shoot to root to maintain root aerobic
respiration.
• Brachiaria adapts to waterlogging by the
development or increase or aerenchyma in root
tissues. Constitutive formation of aerenchyma in B.
humidicola allows immediate adaptation to
waterlogging
• Better adapted genotypes show thicker roots,
greater aerenchyma formation and smaller steles
(conductive tissue)
Drained Waterlogged
B. humidicola Tully
(tolerant)
B. ruziziensis Br 44-02
(sensitive)
• Cross sections taken at
10 cm form the root tip
• Scale = 0.5mm
Aerenchyma
stele

27. Increased suberization of the outer part of the root
(OPR)
B.humidicolaB.ruziziensis
Drained Waterlogged Waterlogged
O2
O2
B. humidicola
B. ruziziensis
O2
O2
O2
O2
AE AE
• Roots with greater aerenchyma formation
and increased suberization of OPR shows
deeper penetration into waterlogged soil

28. LeafsheathInternodeRoot
• Aerenchyma (arrows) formation in shoots (internode
and leaf sheath) and roots
• Continuum of ventilation form shoot to roots
facilitates gas exchange between atmosphere and
rhizosphere
B. humidicola after 21 days of growth
under waterlogging
O2
CH4N2O? C2H4

29. Replacement rooting
Roots produced after waterlogging (white)
• Roots produced before waterlogging (rotten or decaying)
• Brachiaria genotypes without constitutive formation of
aerenchyma in roots depend on the formation of new
roots with aerenchyma for adaptation to waterlogging

30. Replacement rooting
Cayman Mulato II
Decaying
or rotten
roots
Decaying
or rotten
roots
Replacement roots
Cayman

31. • Most of damage (e.g. leaf chlorosis and leaf senescence) occurs between 7 and 14 days
after waterlogging, period where new roots with aerenchyma started to develop.
• After this, aerenchymatous roots confer adaptation to waterlogging.
Screening for waterlogging tolerance in Brachiaria hybrids
1 day of waterlogging treatment 7 days of waterlogging treatment
14 days of waterlogging treatment 21 days of waterlogging treatment

32. Recovery after drought and waterlogging
• B. humidicola responds to accumulated ethylene in waterlogged soil by
internode elongation and hyponastic growth of leaves
• This plastic response allows leaves of B. humidicola to escape form water and
continue photosynthesis
Irrigated Drought Waterlogging Recovery period
Irrigated Drought Waterlogging

33. Conclusions so far…..
• Cayman seems to be a water spender, not a saver. It attempts to
maximize carbon gain (growth) when water is available.
• Most Brachiaria grasses combine water saving mechanisms (by
regulation of water loss by closing leaf stomata) with deep rooting
ability to avoid drought stress.
• Digital images (e.g., RGB, Thermal IR) allows recordings of growth
and responses to stresses.
• Leaf and canopy temperatures could be used as proxies for rooting
depth in Brachiaria genotypes
• Aerenchyma might aid root elongation under drought conditions

34. Conclusions so far….
• Brachiaria grasses adapt to waterlogging by
increasing or developing aerenchyma in roots to
sustain root aerobic respiration
• Plants without constitutive formation of
aerenchyma in roots depend on the formation of
new roots with aerenchyma for adaptation to
waterlogging
• Maximum rooting depth can be used as an
indicator of root aeration efficiency and
waterlogging tolerance

35. Challenges ahead
• Determine the role of endophytes in
drought adaptation
• Determine the contribution of climate
smart Brachiaria grasses to soil carbon
accumulation and greenhouse balance
• Scale up for automated phenotyping in
breeding populations