Plant and Soil 96, 293-296 (1986). Ms.

6482
9 1986 Martinus NijhoffPublishers, Dordrecht. Printed in the Netherlands.

Anatomical, morphological, and physiological responses of alfalfa to flooding

D.M. ZOOK, D.C. ERWIN, and L.H. STOLZY

Department of Biochemistry, Department of Plant Pathology, and Department of Soil and
Environmental Sciences, University of California, Riverside, CA 92521, U.S.A

Received 10 July 1985. Revised May 1986

Key words Aerenchyma Anaerobiosis Ethylene Phytophothora

Summary Flooding of alfalfa plants (3 wk old), resistant (A77-10B) and susceptible (Moapa 69) to
Phytophthora megasperma f. sp., medicaginis (Pmm) for 4-days caused an increase in length (2 cm)
of tap roots, cessation of secondary growth, suppression of lateral root growth, and formation of
aerenchyma channels in the stele and hypertrophied lenticels, which extended to the stele, on
taproots. In soil containing plants from which foliage had been clipped, flooding induced a decrease
in Oz from 21 to 2% by the second day, and caused death of plants. In soil containing intact
unclipped plants flooding induced a slight decrease in 02 to 15 to 17% which returned to normal
by the fourth day.

Introduction
Excess flooding causes severe root injury to alfalfa, Medicago sativa L., especially at high soil
temperature6; flooding also predisposes plants to Phytophthora root rot, caused by Phytophothora
megasperma f. sp. medicaginis referred to hereafter as Pmm) ~~"~3and caused cessation of root and
stem growth for 3 weeks 12.02 deficiency in flooded soils is the primary cause for many adverse effects
such as retardation of shoot growth, premature senescence, mineral deficiency in leaves, and
restriction of root growth 3'4:~ In tobacco 8 and in tomato 9 ethylene accumulation in soil and in
leaves and stems of plants was associated with flooding.
Our recent work indicated that preinoculation flooding reduced resistance of germ plasm
A77-10B and other cultivars of alfalfa to Prom~3.The increase in susceptibility was associated with
concomittant anatomical and physiological changes in the roots and to emanation of ethylene from
foliage of flooded but not unflooded plants. Exposure of the foliage of unflooded plants to ethylene
induced morphological changes in roots similar to those induced by flooding (Zook, Eaks and
Erwin, unpublished~3). We describe herein similar antomical and physiological changes in roots of
a Phytophthora resistant germplasm 'A77-10B '7 and a susceptible cultivar 'Moapa 69' which
followed either a 4-day flooding period or exposure of foliage to ethylene.

Materials and methods

Growth and flooding of alfalfa plants

Seeds, inoculated with Rhizobium meliloti, were planted in steamed U-mix (50% peat moss:50%
fine sand v/v) in 10 • 10 • 10 cm plastic pots in the greenhouse. Plants (3 wk old) were flooded for
4 days in Nalgene wash tubs. One wk after flooding the symptoms of flooding injury and tap root
lengths (200 roots per replicate, 3 replicates) were recorded. Experiments were run three times.

Preparation of roots for observation by scanning electron microscopy (SEM)

Tap roots (5-mm-length) were fixed in aqueous 3% glutaraldehyde (pH 6.8) for 1.5 h, washed
three time (10 min each) in 0.1 M phosphate buffer (pH 6.8), post-fixed in a 0.1 M osmium tetroxide
solution for 1.5 h, washed three times in phosphate buffer, dehydrated in a 30, 50, 70, and 80%
ethanol series for 15 min each, in 90% ethanol for 30 min, in 100% ethanol overnight, critical point
293
294 SHORT COMMUNICATION

dried, coated with gold palladium, and examined with a Jeolco Scanning Electron Microscope
(Model JSM U3).

A,Ieasurement of the physiological responses of roots to flooding

02 in the soil atmosphere was sampled using gas-tight stainless steel collection probes (0.5-ml
capacity) at 5 cm depth 1 day before flooding, each day during the flooding period, and 1 day after
soil was allowed to drain. Samples were removed with a gas chromatography (GC) syringe, and
analyzed by GC. The oxygen diffusion rate (ODR) in the soil was measured with an Oxygen
Diffusion Ratemeter (Jensen Instruments) at 5 cm depth.

Fig. I. Scanning electron micrograph of the stele tissue of Moapa 69 and A77-10B alfalfa roots
(cross sections except D). (A) Unflooded root (60 x ). (13) Roots flooded for 3 days (220 x ) (note
dissolution of parenchyma cells). (C) Roots flooded for 4 days (120 x ) (note dissolution of paren-
chyma cells). (D) Longitudinal section of aerenchyma channels in the stele of roots flooded for 4
days (65 • ). (E) Hypertrophied lenticels formed on the surface of roots flooded for 4 days (65 x ).
(F) Aerenchyma channels in the stele of roots from plants exposed to ethylene (0.1 #1/1) for 3 days
(32 • ). V = vessel; F = fiber; P = parenchyma; ae = aerenchyma; L = lenticel; and ep =
epidermis.
SHORT COMMUNICATION 295

Effect o f ethylene on disease severity on roots

Ethylene (0, 0.01, 0.1, and 1.0 ;d/l) was directed by continuous flow into chambers with open tops
(30 x 30 x 45 cm) containing plants (3 wk old) in 400 ml pots, each of which contained 30 plants.
The ethylene was delivered from an air-ethylene mixing board at a rate of 60 liters per h (monitored
twice daily by GC). The air was purified of background ethylene by passage through a Purafil filter
(Ale2 9KMnO4). Plants were incubated in a growth chamber illuminated with 4-40 watt fluorescent
Westinghous Grow lights. The temperature ranged from 22~ with lights off (12h) to 30~ with
lights on (12h).

Results and discussion

Morphological and anatomical responses to flooding

Flooding for 2 days induced an increase in the length of the tap roots of A77-10B from 6 mm
(unflooded) to 18 mm on day 1, and from 9 mm (unflooded) to 24 on day 2. Similar results occurred
on Moapa 69. Lateral roots did not form on flooded plants. Flooding for 4 days caused stunting,
chlorosis of foliage and necrosis of the xylem tissue in roots. In flooded soil root hairs and Rhizobium
meliloti nodules formed directly on tap roots rather than on laterals. Flooded plants remained
stunted up to 10wk after flooding.
The internal anatomical response of roots to flooding was similar for both A77-10B and Moapa
69 plants. In unflooded roots the central vascular stele bounded by the endodermis, the cortex, and
the epidermis were well defined (Fig. 1A); however, after 3 days of flooding, dissolution of the
parenchyma cells in the stele was observed (Fig. 1B), but there was no evidence of plugging of
vessels. After 4 days of flooding, cavities (aerenchyma tissue) formed in the stele, which appeared
to be due to complete dissolution of the parenchyma cell walls 5 (Fig. 1C). Aerenchyma extended
throughout the length of the tap root (Fig. 1D). Hypertrophied lenticels, which extended from the
epidermis to the stele, were also observed 4 days after flooding (Fig. 1E).
Ethylene at 0.01, 0.1 and 1.0#l/liter, applied to the foliage of intact A77-10B and Moapa 69
plants, induced anatomical effect on roots similar to those described above for flooding, however,
aerenchyma induced by ethylene (Fig. IF) occurred one day sooner. Production of aerenchyma in
other plants has also been associated with ethylene3'5, however formation of aerenchyma in xylem
tissue is rare 3.

I -- I I 1 I I ' M-6" = i i i i i
A 7 7 - lOB
2O

15
9 + Fol no flood
% o2 o-Fol no flood
io
A + F o I flood
9 - Fol f l o o d
5

k .
o , , , , T - T - - r i i i i "~-t-'~,
-24 o +24 + , 8 ~2 +96 +,2o -24 0 +24 +48 +72 +96 +120

Flooded Dreined Flooded Droined

TIME (h) TiME (h)

Fig. 2. Effect of a 4-day flooding period on the concentration of 02 (analyzed by gas chromato-
graphy) in the soil atmosphere in which clipped and intact plant (3 wk old) of the alfalfa cultivars
A77-10B and Moapa 69 (M69) plants were growing in steamed UC mix. Treatments: foliage (stems
and leaves) removed before flooding ( - fol flood); foliage not removed before flooding ( + fol
flood); foliage removed and not flooded ( - fol no flood); and foliage not removed before flooding
( + fol no flood).
296 SHORT COMMUNICATION

Physiological responses of plants to flooding

In several experiments with A77-10B plants flooding caused a decrease in the ODR from
0.66 #g/cm2/min 24 h before flooding to 0.11 #g/cm2/min 24 hr after flooding; however, 48 hr after
flooding the ODR increased to 0.34#g/cm2/min. The effects were similar on Moapa 69 plants.
Flooding decreased soil 02 (measured by GC) from 20.3% 24 hr before flooding A77-10B plants to
18.2% 24 hr after flooding; however, after 48 hr of flooding 02 increased to 20.1% and remained at
that level for the duration of the experiment. The effects on Moapa 69 plants were similar.
Since 02 diffuses downward in flooded roots most likely via aerenchyma3, the effect of clipping
and removal of stems and leaves prior to flooding on soil 02 was determined. In soil containing
clipped plants, 02 dropped from 21.8% 1 day before flooding to 0.5% 48hr after flooding and
remained at that level for the 96 hr flooding period; however, in flooded soil containing intact plants,
02 decreased from 21.4% to about 16-17% 24 hrs after flooding and increased gradually to normal
levels 96 hr later (Fig. 2). Because removal of foliage markedly increased the adverse effect of
flooding, translocation of 02 from the foliage to roots appears to be necessary for survival of alfalfa
in flooded soil. A similar response of clipped plants to flooded soil at high temperatures occurred
in field experiments6.
The formation of aerenchyma tissue in the stele of flooded alfalfa roots has not been previously
reported. Aerenchyma channels are considered to be a means of transport of 02 from the leaves to
roots in flooded soils3'5'~~ Since ethylene biosynthesis in flooded alfalfa plants initiated formation
of aerenchyma tissue (Zook, Eaks and Erwin, unpublished~3), this mechanism could effect moderate
flood tolerance in alfalfa by allowing increased translocation of oxygen to flooded roots. However,
these morphological changes also render the plant more susceptible to Phytophthora root rot (13;
Zook and Erwin, unpublished~3). These data contribute in part to an explanation of predisposition
of alfalfa roots to Phytophthora root rot T M and the "wet soil syndrome" of alfalfa 1.

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