The Use of a Multi-Celled Apparatus
for Anaerohie Studies
of Flooded Root Systems 1
D. L. Culbert and H. W. Ford 2
Agricultural Research and Education Center, Lake Alfred
Abstract. A circulating system with
sufficient versatility to permit monitoring and
control of solutions in the flooded root
rhizosphere is described. The system
circulates anaerobic solutions around plant
roots or can be used to simulate the type of
flooded environment necessary for anaerobic
bacterial activity. The composition of the
solution can be monitored before, during, and
after contact with the roots. Oxygen
deficiency per se was relatively harmless to
citrus roots, whereas severe root damage
occurred from exposure to < 3 ppm total
sulfides a: pH 6 for 7 days. Tolerance to
nooding of rough lemon appears to be
associated with tolerance to injury by H25.
Investigations by Ford (2, 3, 4, 5)
indicate that bacterially generated
sulfides during anaerobiosis are one of
the principal causes of citrus root
damage under oxygen deficient flooded
conditions. The determination of toxic
levels of soluble sulfide as influenced by
soil pH, temperature, root density, and
certain cations could not be answered
by flooding plants in containers. A.
method was req uired that would permit
separation of ~-he factors found in
flooded soil.
........,.,.. .
A multi-unit system was constructed
tha t can circula te anaerobic solutions
around seedling roots or be used to
initiate the type of flooded environment
necessary for anaerobic bacterial
activity. Composition of the solution
can be monitored before, during, and
after flowing over the plant roots.
Changes in composition of the solution
can be made during circulation. Once
the desired environment has been
established, circulation of solution can
be stopped and the rate of bacterial
ana erobiosis measured at periodic
intervals for the zone abutting the
feeder robt system.
This study was limited to the effects
of H2S, but the circulating system is
adaptable to other plants and anaerobic
problems.
lR.eceived for publicution July 26,1971.
Florida Agricultural Experiment Stutions
)ournul Series No. 3973. Contribution from
Soil-Wuter-Atmosphere-I'bnt (SWAI') Project
in coopcrution with the Southern Brunch. Soil
und Wuter Conservution Research Division,
Agricultural
Research
Service.
U. S.
Department of Agriculture.
lCruduate
student and
Horticulturist,
respedi\'ely, Univer;;ry of Florida. IFAS.
Design of the apparatus
The system is capable of handling 2
solution treatments of 6 replications or,
if desired, a single solution treatment of
12 plants involving such subtreatments
as cultivars, root density, etc. The use of
rubber tubing and stoppers should be
held to a minimum because of porosity
to H2S. Coating with paraffin and other
agents was not worth the effort.
"Tygon" is preferred over compounded
rub ber for coupling glass tubing.
The apparatus is illustrated in Fig. I
and 2. Solutions are prepared in two 21
liter carboys (A, B) and circulate from
each carboy to the base of six 250 ml
separatory funnels (C), over the root
system (0) in the washed sand filled
funnels, through a 12-line Ourrum
dial-a-pump (0), and back to the
carboy. The pump was adjusted to a
flow rate of 7 mljmin per line. By
manipula ting 2 straight bore stopcocks
(E) and four 3-way stopcocks (F),
solutions can be circulated from 1
carboy to 6 or 12 funnels or from 2
carboys to 6 or 12 funnels.
A l·liter Sq uib b pear-shaped
separatory funnel (G) replaces solution
lost from sampling and transpiration.
The reserve solution exerts a slight
pressure preventing air from entering
the system. An 02 trap (H) containing
5 % a lkaline potassium pyrogallate
prevents 02 from entering the reserve
solution.
Four sampling locations are
provid ed: a thistle tube port before the
solution enters the funnels (I); a tube
terminating in the center of each funnel
abutting feeder roots (1); a thistle tube
port after the solution leaves the funnel
(K); and directly from the carboy (L).
The thistle tube ports are designed
specifically for membrane type 02
electrodes (M). Because measurements
are usually for 02 values below 0.2
ppm, the electrode is calibrated to zero
by flushing the thistle tube with N2
before flooding the membrane with the
circulating solution. Continuous
monitoring with an 02 electrode can be
accomplished by removing the thistle
tubes from ports I and K and mounting
the electrode directly in the flow line
Seedlings are planted in the funnels
in washed, graded, sterile, white sand.
Each funnel is filled 3/4 full of solution
to be circulated. A 3-cm diam fiberglass
disc (N) cut from porous fiberglass
HORTSCJ[~CE. VOL 7(l L FEBRUARY 1972
sheeting is placed in the bottom and an
inch of dry sand poured into the funnel
to'seat the disc. The root system (0)
and sampling tube (1) are suspended in a
funnel and sand poured around them. A
constant solution level is maintained
during sand filling by slowly draining
solution from the bottom of the funnel
through a stopcock (P).
A no. 11 two-hole rubber stopper
(R) was cut to insert the seedling(s) as
well as support the exhaust line (T) and
a solution sampling port (1), is inserted
in the top of the funnel. The crown area
of the seedling and the slit in the
stopper are sealed with grafting wax.
Four hundred ml of solution, identical
in composition to the solution in the
carboy, is flushed through each funnel
before circulation is started to remove
trapped 02 from around the roots.
Ox y g en-deficient solutions were
prepared by bubbling N2 into the
carboys, through a gas dispersion tube
(V), for 24 hl' prior to circulation. A
low redox, anaerobic environment can
also be established in 24 hr by
circulating distilled water containing 3
ppm of H2S for 8 hr followed by an
additional 2 ppm for 16 hL The use of
H 2 S is recommended for studies
involving initially low redox values
necessary to stimulate development of
such organisms as the sulfate reducing
bacteria. Soluble sulfides can be
injurious to roots at low concn with
prolonged exposure; toxic levels should
therefore be esta blished for specific
cultivars.
A concd solution containing 3,200
ppm of total sulfides was prepared by
saturating 0.1 N NaOH with gaseous
H2S at 25 0 C in an apparatus shown in
Fig. 3. Such solutions should be
prepared under adequate ventilation and
stringent safety precautions. Sulfide
saturation was complete when PbS
formed in the lead acetate trap located
in the exhaust line. The potassium
py rogalla te trap prevented 02
contamination when the above solution
saturated with H2S was removed from
the burette. Calibrated amounts of this
solution were drained directly from the
burette into a carboy or transferred
from the burette to the carboy with a
50 ml calibrated glass syringe (a small
bore polyvinyl chloride tube was
substituted for the metal needle).
Sulfide content of diluted H2S
solutions, as prepared in carboys, was
determined colorimetrically by the
p-aminodimethylaniline method (6)
Soluble sulfide as a citrus root toxicant
Floodin~ injury from 7 days vi 02
deficiency compared to that ii'om
sulfide. The solution and components of
the circulating system were initially
aseptic. All lines were flushed with
alcohol, the sand was steamed. and the
29
H
N-IUtlt>,
p
I
Fig. 1. Schelnatic view of so!ulioll-cil'culating J.ppura£us.
A. 21 liter carboy
H. Oxygen traps.
B. 21 liter carboy
I. Sampling port (before fUllnels)
C. 250 ml separatory funnels
.I
Sampling tube in center uf funnel
D. 12-line circulating pump
K Sampling port (after funnels)
E. Straight bore stopcocks.
L Sampling line in center of carboy
F. 3-way stopcocks.
M. Oxygen electrode.
G. Peur-sh aped sepa ra tory fU nnels_
N J:iberglass disc.
O. Root system.
P. Fill und drain stopcock.
R. No. I I stopper.
S. Slit in stopper.
T. Exhaust line (funnnllO pump).
V. Gas disper:;ion tube.
-.
250mL
burette
I
K pyrogallote
dispersion'·
tube
Fig.
Fig. ] The ')olutiun
30
c.:irt:ul~ting. appfHaTus
3
Apparatus for saturating a solution
with H2S
HORTSC1fNCE VOL 7(1),FEflRUARY 1972
glass distilled water handled aseptically.
Only roots of the 12 six-month-old
rough lemon seedlings were septic.
Oxygen deficient distilled water was
circulated over the roots of 6 seedlings
at 25.6 0 C at pH 6.2 for 7 days. The 02
deficient level was prepared by bubbling
N7 into 21 liters of distilled water for
24hr prior to clrculation. A slow rate of
N7 bubbling was continued during the 7
da-ys of circulation to hold the 02 level
to zero and to remove soluble sulfide in
the root rhizosphere that might be
generated by bacterial action.
The root systems of the remaining 6
plants were flooded with a solution
maintained at 2.5 ± 0.3 ppm total
sulfides adjusted to pH 6.2 for a period
of 7 days. Zero 02 occurred within 6 hr
after initial circulation.
The plants were removed from the
funnels after 7 days of flooding,
transplanted to well-drained sand, and
IH.:ld for 10 days to note persistence of
root injury and plant survival.
None of the plants (02 deficient or
s u bj e cted to sulfide) showed
pronounced epinastic sympto ms during
the 7 days in the circulating apparatus.
Root systems of plants from the 02
deficient treatment showed no visible
injury when they were removed from
the funnels. New root growth from ta p
roots, secondary roots, and feeder roots
occurred ra pid ly during the 10-day
holding period in drained sand. In
contrast, plants that received the 2.5
p pm sulfide treatment for 7 days
showed visible injury to feeder roots
characterized bY-wat.er soaled areas and
cortex sloughing. After 10 days in
aerated soil, new growth occurred only
from the upper 50% of tap roots and
from a few secondary roots near the
crown. All the original feeder roots,
most tap roots, and secondary roots
were Jdlled.
A longitudinal strip of wood from
the tap root was removed from selected
seedlings (prior to transplanting into
aerated sand and after completion of
the 7-day flooding test). The section of
wood removed contained phloem,
cambium, and xylem. The segment of
wood was stained with 0.1% solution of
p-ami nodimethyla niline plus 10% FeCl3
by the method of Takagi (7) to note the
presence of sulfide in tissues. Stained
tap root segments showed sulfide concn
as methylene blue areas. Dark blue areas
in phloem and xylem correlated with
the regions of the tap root that were
dead at the end of 10 days in aerated
sand.
These studies indicate that 02
deficiency per se was relatively harmless
to citrus roots during 7 days of
flooding, whereas severe root damage
occurred from 2.5-3.0 ppm of soluble
sulfide at pH 6 in the root rhizosphere.
There was insignificant root da mage in a
similar experiment involving
2 ppm
H2S in the circulating solution.
Tolerance of 3 cultivars to soluble
suUide. Six-months-old seedlings of
rough lemon, sour orange, and
'Cleopatra' mandarin were flooded for 6
days with a solution containing 3 ppm
total sulfides at pH 6.0. At the end of 6
days of circulation, the feeder roots of
aU 3 cultivars were killed as indicated by
water soaking and cortex sloughing. In
addition, a substantial number of
secondary roots on the sour orange and
'Cleopa tra' mandarin seedlings were
killed as indicated by sloughing of the
<
Field Testing of Weak Acids
for Facilitating Citrus Fruit Harvest
under Florida Conditions I
W. C. Wilson 2
Agricultural Research and Education Center,
Lake A Ifred, Florida
Abstract. Various weak acids produce
citrus fruit abscission in Florida. Ery thorbic
(ascorbic) acid (1-2%) or hexamic acid (1-2%),
alone or combined with citric acid (total
concn 1-2%), produced acceptable abscission
I Received for publkation September 27,
1971.
Florida Agricultural Experiment
Stations Journal Series No. 4077.
2 Assistant
Plant
Physiologist.
Florida
Department of Citrus. University of Florida.
IFAS. The author wishes to acknowledge the
te~hnical assistan~e of WIr. C. J. Johnson and
Mr. E. H. Rowland .
. .-
but only of early and midseason orange~ for
cannery use. Pho~phoric acid (1/2%) and
ferric chloride (0.5-2_5%) produced erratic
loosening, phytotoxicity, severe peel injury,
and sometimes damaged spray equipment.
'ne type of peel injury resulting from weak
acid sprays may cause e:o.:tensive rotting (40%
or more) if extended periods of wet weather
occur prior to picking. As weather forecasting
is not sufficiently accurate to predict
conditions more than 48 hr in advance, and
fruit abscission normally occurs from 3-7 days
foUo,,~ng spraying, the periodic economic
losses IIlat could be e:o.:pected under these
conditions preclude their use in Florida.
HORTSCIENCE. VOL. 7(l),FEBRUARY 1972
root cortex. When the plants were
transplanted to aerated soil for J 2 days,
all 'Cleopatra' and sour orange seedlings
died. Rough lemon seedlings survived
and new feeder root growth was
initiated from tap roots and some
secondary roots. The results agree with
relatively broad spectrum flooding
studies involving plants in containers of
soil (3) where the citrus root systems
were merely flooded with water for 2
weeks or longer. In these studies, rough
lemon ranked high in survival following
flooding. Tolerance to flooding of rough
lemon may be associated with tolerance
to sulfide injury by H2S or to its ability
to regenera te a functional root system
more rapidly than the other 2 cultivars.
Li tera ture Ci ted
I. Connell, W. E., and w. H. Patrick. 1968.
Sulfate reduction in soil; Effects of redox
potential and pH. Science I S9;86-87.
2. Ford, H. W. 1965. Bacterial metabolites
that affect citrus root survival in soils
subject to floodings. PYoc. Amer. Soc.
Hart. Sci. 86;205-212.
3.
. 1969. Water management 01"
wetland citrus in 1;lorida. Proc. 1st 1ntl.
Citrus Symp. 3; 1759-1770.
4.
, and O. V. Calvert. 1966.
Induced anaerobiosis caused by flood
irrigation with water containing sulfides.
Proc. Fla. State Hart. Soc. 79; 106-109.
5.
and
1967.
. Relationship between excessive soil
moisture and citrus root damage. Fla. Agr.
Expt. Stu. Ann. Rpt. State Project 1281.
1967;247.
.
6. Standard Methods for the Examination of
Water, Sewage and Industrial Wastes.
1955. lOth t:d. Amer. Pub. Health Assoc.
New Yl.rk.
7. Takagi. S., and H. Okajima. 1956.
Physiological hehavior 01" hydrogen
sulfide, Part 3. Detection of sulfide in the
rice plant. Sci. Rep. Res. inst. TO!lOku
Univ. I)·Vol. 7(t);17-26.
Sprays of various weak acids have
been tried for inducing abscission (fruit
loosening or pull-force reduction) of
Florida citrus fruits during the past
several years. Ascorbic and erythorbic
(EA) acids combined with citric acid
(CA) (2, 6), hexamic acid (6), and
various ferric and co pper-containing
compounds (I) have been proposed as
possible abscission agents. In some
cases, sizeable tests have been applied,
with generally good results on early and
midseason oranges, but not on late or
'Valencia' oranges (6). Leaf drop has
rarely been a problem with EA + CA or
hexamic acid, though occasionally
erratic results in fruit loosening were
observed (7). Rains soon after spraying
usually resulted in no fruit loosening
(8). As prospects for "label approval"
appeared reasonably good after 2
complete seasons of testing (6).
attempts were made during the 1969-70
fruit season. and again in the 1970-71
frujt season, to make large-scale picking
trials with these compounds.
EA + CA was applied to 'Hamlin' and
_~
1