Ihysiological

Plant Pathology

( 1985) 26, 125-l

34

Ethylene biosynthesis and related physiological changes

in Penicillium digitatum-infected
grapefruit (Citrus
para disi) t
0.

IZCHILEA,

Department
Irrarl

E. CHALUTZ~,

of Frutt

i.lrcept~d,fbrpublicati~n

Biochemical
grapefruit

Y.

Vegetable Storage,

FUCHS

and I. ROT

Agricultural

Research

Organization,

The

Volcani

Center,

Bet Dagan

50250,

September 19841

and physiological

changes and their relation to ethylene biosynthesis

were studied in
cv. Marsh Seedless) peel, 556 days after inoculation
with Penicilalbedo and flavedo tissues of the peel, fungal invasion was associated with increases in free galacturonic
acid but with reductions
in pH and soluble proteins. The
rxtrnt of the changes was smaller the greater the distance from the maceration
front. Two parallel
and distinct maceration
fronts could be defined in the peel, the one in the albedo preceding
that in
the llavedo. Fungal glucosamine
was present in the apparently
healthy region of the albedo up to
15 mm ahead of the flavedo maceration
front.
Fungal invasion
was associated
with increases in both l-aminocycloproprane-1-carboxylic
acid (ACC)
and ethylene
production
but the ability of the tissue to convert ACC to ethylene
decreased with the development
of the infection. The early relatively
low rate of ethylene production in infected fruit seems to originate
mostly from the fruit tissue while a later and higher rate of
rthvlene
production
originates
mostly from the fungus.
(Citrus paradisi Macf.
liurn digitatum Sacc. In both the

INTRODUCTION
Citrus fruits are usually classified as nonclimacteric
fruits due to the fact that mature
fruits do not exhibit a rise in either respiration
or ethylene production
through
their
normal pre- and post-harvest
life [6, ,281. H owever, when fruits are infected by the common postharvest
pathogen
Penicillium digitatum Sacc., ethylene is produced
rapidly
[IO,
271.
Increased ethylene production
is a common feature of diseased plants [22,29] and
while ethylene may originate
from the fungus, and in fact P. digitatum can produce it in
culture [II, 171, it may also be produced
partly or entirely by the plant as a response to
infection
[12]. Stress conditions
have been reported to increase the levels of l-aminocyclopropane1-caboxylic
acid (ACC), the immediate
precursor of ethylene in higher
plants [I]. Generally,
a lag period is observed between increases in ACC levels and subsequent increases in the rate of ethylene production
[14, 161. However, in several plant
N~ontribution
from the Agricultural
Research Organization,
No. 793-E 1983 series.
$I0 whom all correspondence
should be addressed.
Abbreviations
used in text: ACC, l-aminocyclopropane-1-carboxylic
ofthe maceration
front; MF, maceration
front, at edge ofM2;
MZ,
green spore zone; WMZ, white mycelium
zone surrounding
SZ.
0048-4059/85/020125+

10 $03.00/O

The

Volcani

acid;
maceration

1985 Academic

Center,

Bet Dagan,

HZ, symptomless
zone surrounding
Press Inc.

(London)

Israel,

zone ahead
WMZ;
SZ,
Limited

126

0. Achilea

et al.

systems, for example post-climacteric

avocado fruit [14], accumulation
of ACC is not
always followed
by a marked
increase
in ethylene
production.
Although
some
micro-organisms
can utilize ACC as a nitrogen source [15J, it is not clear whether P.
digitatum can utilize ACC for ethylene production.
During
infection
of citrus fruits, P. digitatum produces pectolytic
enzymes one of
which has been characterized
as an exo-polygalacturonase
(poly- 1,4-D-galacturonide
glycanohydrolase,
E.C. 3.2.1.15)
[4, 5]. Th is enzyme hydrolyses
cell wall pectin to
D-galacturonic
acid monomers,
which results in a loss of physical stability of the infected tissue. n-galacturonic
acid in turn causes plasmolysis
of peel cells and swelling of
their walls leading eventually
to tissue maceration
[5j. Barmqre
& Brown [5j found a
maximal
level of 12 mg g- of D-galacturonic
acid in peel in P. digitatum infected
Hamlin
and Valencia
oranges, while Miyakawa
[ZO] detected a level of more than
in Satsuma oranges during the early stages of infection with P. digitatum.
20mgg-
We report here the results of a study on some physiological
changes which occur in
P. digitatum-infected
grapefruit
and the relationship
of these changes to ethylene
production.
MATERIALS

AND METHODS

Plant andfungal material

Grapefruits
(Citrus paradisi Macf. cv. Marsh Seedless) were harvested in mid-season,
and stored at 11-14C
in 8O-9Oo/o RH until use. For inoculations,
a single-spore
culture of P. digitatum Sacc. (ATCC No. 10030) was used. The culture was propagated
on potato dextrose agar slopes. Prior to inoculation,
the fruits were washed with water
and then surface sterilized with 70% ethanol. They were inoculated
20 mm away from
the stem end by introducing
a 5 x 5 x 1 mm block of agar infested with mycelium into a
V-shaped
cut, 3 mm deep. The wound was then covered with masking tape, and the
fruit incubated
in the dark at 20-23 C in a ventilated
chamber.
For the various tests, samples of peel were removed with a scalpel (4 mm wide
strips) or with a cork borer (12 mm diameter),
and the albedo and the flavedo tissues
separated.
Representative
samples were taken from several defined
regions of the
inoculated
fruit [Fig. 1 (a) 1. A minimum
of nine fruits was used for each assay.
Six days after inoculation,
several, well defined zones could be observed on the fruit
surface. These were defined as follows [see Fig. 1 (a)]: green spore zone (SZ), a zone 50
to 80 mm in diameter
around the inoculation
site, where mature green conidia had
developed;
white mycelium
zone (WMZ),
which consisted of an area approximately
13 mm wide, surrounding
the SZ and characterized
by dense white mycelium;
maceration zone (MZ), which consisted of a belt approximately
17 mm wide surrounding
the
WMZ and characterized
by the soft peel, and the indistinct
appearance
of oil glands on
its surface; healthy areas (HZ), consisting of the rest of the fruit, where no infection
symptoms were visible; the maceration
front (MF), visible as a distinct line between the
MZ and the HZ.
Since the distance of any part of the peel from the MF was found to be a useful
reference point for measuring
the extent of fungal invasion
and the extent of any
physiological
changes taking place in the infected fruit, all results were expressed in
relation
to this distance [Fig. 1 (a)]. Sites in the HZ were given positive values, while
sites in the infected zones were given negative values.

Biochemical-physiological

changes

in infected

grapefruit

127

Biochemical andphysiological
assays
The amounts of fungal mycelium
and spores in infected tissues were determined
by
determining
the amount of glucosamine
present since glucosamine
is a component
of
the fungal cell wall which is not found in higher plants [13]. The sensitivity
of the
method of Netzer et al. [21] was enhanced
by extending
the hydrolysis
time from 6 to
9 h.
The rate of degradation
of pectic substances in the peel was determined
by an assay
for o-galacturonic
acid [7]. One gram of tissue was homogenized
with 10 ml of 700,,,
ethanol at 4 C for 1 min using an Ultra-Turrax
macerator.
The homogenate
was then
centrifuged
at 27000g
for 10min
at 4C and the supernatant
used for the Dgalacturonic
acid analysis. In the same way water extracts were prepared of the albedo
and flavedo for the determination
ofpH, soluble protein [S] and ACC content [18].
Production
of ethylene by peel tissues was determined
by the following
method.
Samples, each of five peel discs, were weighed and placed inside 2 1 ml glass vials. One
hour later, the vials were sealed with rubber serum caps and incubated
for 60 min at
24 C in the dark. A 2 ml sample of the atmosphere
above the discs was then withdrawn
by a hypodermic
syringe, and its ethylene content determined
using a Packard gas
chromatograph
equipped
with an alumina
column and a flame ionization
detector.
Under these conditions,
production
ofwound-induced
ethylene could not be detected.
In some experiments,
the excised discs in the vials were incubated
for periods of up
to 32 h and ethylene accumulation
was determined
during 1 h periods, 2,8, 16, 24 and
32 h after excision. After each 1 h sampling
period the vials were aerated before
reclosing.
All results are expressed on a fresh weight basis, except those for glucosamine
content which are expressed on a dry weight basis.
RESULTS
Fungal colonization of the peel
The distribution
of fungal mycelium,
as determined
by the glucosamine
assay, in the
various regions of the fruit peel is shown in Fig. 1 (b). The fungus was found not only in
the zones showing symptoms, but also in adjacent apparently
healthy tissue. The total
content of the fungus in the flavedo decreased
with increasing
distance from the
inoculation
site, but small amounts of glucosamine
were found as far as + 15 mm away
from the MF in the flavedo of the HZ. Glucosamine
in the albedo decreased with
increasing distance from the inoculation
site just as in the flavedo but the amounts were
considerably
less. The differences were most obvious in the WMZ and in the SZ. The
most distant site in the albedo where glucosamine
could be detected in the apparently
healthy zone was at + 5 mm.
Physiological changes associated with infection
Fungal infection markedly
affected D-galacturonic
acid content in the peel [Fig. 1 (c)].
Healthy
albedo and flavedo tissues contained
only l-2 mgg-
of free galacturonic
acid, but sites closer to the MF than + 10 mm in the albedo or +5 mm in the flavedo
contained
up to 14 mg g- in the MZ and the WMZ, with a slight decrease in the SZ of
the albedo. Galacturonic
acid content was generally
higher in the albedo than in the
Havedo of macerated
regions. Microscopic
examinations
of the MF region showed that

Stem end
of the fruit
\

\
\\

(b)

284Q412 a4-

-I=*-

Ot 70

DMmce

-..&
III1

t20tlO

-------

---------I

0 -10 -25

I
-50

?j
I
-70

I front of (t 1or behind (-) macemtlon front

(mm1

FIG. 1. Biochemical
and physiological
changes in the peel of P. digitatum infected grapefruit
in
relation
to ACC content
and ethylene
production.
(a) Diagram
of an infected grapefruit
6 days
after inoculation
illustrating
the zones of tissue analysed
in relation
to the maceration
front: HZ
symptomless
zone ahead of maceration
front; MF, maceration
front;
MZ, maceration
zone;
WMZ,
white mycelium
zone; SZ, green spore zone. (b)-(g)
Ch an g es in biochemical
and physiological
parameters,
in infected
fruits in relation
to distance
from the maceration
front.
(b)
Glucosamine
content.
(c) Galacturonic
acid. (d) pH of water extract.
(e) Water soluble protein
content.
(f, ACC content.
(g) Ethylene
production.
All assays were carried
out on peel tissue samples taken from healthy
or P. digitatum infected
grapefruits,
6 days after inoculation
near the stem end of the fruit. The fruits were incubated
at
21 C in the dark. Assay procedures
are given in the Materials
and Methods
section. Bars indicate
SD. O-0,
Healthy
fruit albedo;
a----a,
healthy
fruit flavedo;
0~ -----0,
infected
fruit
albedo; A----A,
infected fruit Bavedo.

Biochemical-physiological

changes

in infected
TABLE

77~ e&cl

of adjusting

thepH

Non-treated
(mg g- I flwt)

Albrdo

1.87 + 0.062

k'lavrdo

3.15*0,073

soluble

129

ofthe water extracts

Water
Peel
I issur

grapefruit
ofpeel on soluble prolein contenl

proteins

in the extract

Adjusted to pH 4.5
(<> ofnon-treated)
82.4k6.6
80.8f3.9

Adjusted
to pH 3.5
I,,, ofnon-treated)
80.0f4.6
75.9 f 5.9

Water extracts
were prepared
from peel tissues of healthy
grapefruits.
extracts was either unchanged
or adjusted after extraction
to pH 4.5 or 3.5
extracts were then incubated
at 0 C for 8 h and water-soluble
proteins were
the method ofBradford
[S]. The original pHs of the extracts of the albedo and
5.2 kO.05 and 5.4 kO.06, respectively.
Each value is a mean of nine replicates

The pH of the
using HCl. The
determined
by
the flavedo were
+ SD.

the albedo tissue was already macerated

underneath
the MF in the flavedo. Thus, two
parallel
maceration
fronts exist with the inner albedo front preceding
the external
flavedo front by 5-10 mm.
Considerable
changes occurred
in the pH of the peel, as a result of infection
as
shown by the pH of water extracts [Fig. 1 (d)]. No significant
differences were found
between the pHs of water extracts from albedo tissues of healthy fruit and those from
healthy zones of infected fruit. However,
a sharp fall in pH, from 5.1 to 3.7 occurred
across the MF within the region of + 20 to - 8 mm. No further reduction
in pH was
found in the infected region. A similar pattern of pH changes was found in flavedo
tissues.
The content of water-soluble
protein in the peel of healthy and P. digitatum infected
grapefruit
is shown in Fig. 1 (e). In healthy fruit, there is a small but steady decrease in
the protein content of the flavedo tissue with increasing
distance from the stem end of
the fruit. In P. digitatum infected fruit, however, there is a sharp decline in water-soluble
from +20 to -8 mm from
proteins, from 4.1 1 to 0.73 mg g- , in the region extending
the 14F. The same low value was maintained
in the whole infected region. A similar
pattern of changes of water-soluble
proteins was found in the albedo tissues but all the
values (except in the region -8 to - 70 mm in the infected fruit) were substantially
lower. A constant level of soluble proteins was maintained
in the albedo tissue of the
healthy fruit in all regions.
To determine
whether the reduction
in water-soluble
proteins in the water extracts
[Fig. I (e)] was related to the lower pH of the water extracts [Fig. 1 (d)], we examined
the eflrct ofadjusting
the pH of the water extracts of peel from non-infected
fruit on its
protein content (Table
1). Reducing
the pH from 5.4 to 4.5 caused a reduction
in
water-soluble
protein content of about 20, but a further reduction
to pH 3.5 had no
additional
effect on protein solubility.
Thus, the reduction
in soluble proteins observed
in Fig. 1 (e), could be accounted
for by the reduction
in pH in the infected tissues at
least to pH 4.5.
Ethylene
production and the infection process
The efrect of fungal infection
on ACC content of the peel is shown in Fig. 1 (f). The
basal level of ACC in healthy fruit as well as in the distal parts of the HZ of an infected

et a/.
fruit was low (0.27-0.32 nmol g-r). The ACC content increased in both albedo and
flavedo during tissue infection. The increase occurred first in the albedo where it
reached a level of 8 nmol g- at +8 mm, and 23 nmol g- at - 25 mm, as compared
with 3 and 29 nmol g- at the same sites, respectively, in the flavedo. The highest
amounts were found in both tissues at the center of the SZ.
The rate of ethylene production by healthy albedo and flavedo discs, shortly after
excision from the fruit, was very low [O-5 nl g- h- , Fig. 1 (g)] and this level was also
found in albedo discs from the infected zones. However, in the flavedo a small but clear
peak in ethylene production occurred in the HZ near the MF with markedly higher
production from discs from the WMZ reaching a maximum of 18 nl g- h- at the
center of the SZ [Fig. 1(g)].
A further study was carried out to characterize ethylene production patterns in
infected fruits in more detail (Fig. 2). Production was followed over a 32 h period following excision and the results indicated similar changes with time in both the albedo
and the flavedo, when they were infected to similar levels. However, the rate of
ethylene production by infected flavedo discs was always higher than that by infected
albedo discs [Fig. 2(a)-(d)]. Rates of ethylene production changed similarly throughout the incubation time in the +70 mm discs from an infected fruit or from the
respective site from a healthy fruit [Fig. 2(a)]. It increased in the albedo discs from 0.3
to 26 nl g - h- during the first 24 h of incubation but then decreased to 2.2 nl g - h- .
This pattern was found to be typical for both albedo and flavedo tissues. A similar
pattern of ethylene production was found in flavedo discs from infected fruit cut at
+8 mm [Fig. 2(b)] but not from albedo discs, which produced ethylene at the low rate
of 3.6 nl g - h- or lower throughout the incubation period. A very low rate of production (0.6 nl g-h- ) was typical for discs of albedo or flavedo cut at - 8 mm in the zone
infected during the first 16 h after excision [Fig. 2(c)], During the following 16 h a
moderate increase in ethylene production occurred from the albedo but a large increase
occurred from the flavedo. Ethylene production by albedo discs from the - 70 mm
region in infected fruit increased rapidly from 18 to 120 nl g- h- during the 32 h
following excision [Fig. 2(d)]. D ense mycelium and spores developed on the surface of
- 70 mm discs of both tissues during the incubation period.
The ability of the fruit peel tissue to convert exogenously added ACC to ethylene
was tested in discs cut from different zones in infected fruit (Table 2). Ethylene production by ACC-treated discs cut from the HZ ( + 70 mm) increased nearly lo-fold while
ACC-treated discs cut from the SZ ( - 70 mm) or from the MZ (- 8 mm) did not produce ethylene at a greater rate than control discs. The ACC was readily taken up by all
discs (see footnote to Table 2).

130

0. Achilea

DISCUSSION

These results clearly show that fungal mycelium is present not only in the macerated
regions of the peel of P. digitatum infected grapefruit, but also in the regions where no
symptoms are visible. The slightly higher glucosamine content of the flavedo, as compared to the albedo tissue in the HZ indicates that fungal mycelium advances slightly
faster in the flavedo than in the albedo. Autolysis of mature mycelium [23,24,2s] could
account for the slight decrease in glucosamine content in the albedo in the SZ.

Biochemical-physiological

changes

in infected

fi
_

02

(cl

16

24

Time

after

131

grapefruit

32

(d)

excwon

16

24

32

of discs (h)

FIG. 2. Time-course
ofethylene
production
by albedo and flavedo discs, cut from different sites
on healthy or P. digitatum-infected
grapefruit.
Ethylene production
by peel discs cut from different
sites of the fruit, as defined in Fig. l(a): (a) + 70 mm; (b) +8 mm; (c) -8 mm and (d) -70 mm.
Cl
0, Healthy fruit albedo; a----n,
healthy fruit Aavedo; O--O,
infected fruit albedo;
A ~~ -A, infected fruit flawdo.
Five peel discs, 12 mm in diameter,
were excised from each site on each of nine fruits. The discs
were separated
into albedo and flawdo.
weighed and placed in 21 ml glass vials. The vials were
incubated
in the dark at 24 C. Accumulation
of ethylene during 1 h periods was determined
at
various times up to 32 h after excision. After each 1 h sample period the vials were aerated before
reclosure.
The data obtained
from healthy control discs are not given for Fig. 2(b), (c) and (d),
since they were similar to those shown in Fig. 2(a). Vertical
bars indicate SD.

The marked increase of o-galacturonic

acid [Fig. 1 (c)] is probably
due to fungal
attack, and results from the hydrolysis
of host pectic substances by fungal exopolygalacturonase
[4,5]. Increases in galacturonic
acid in the infected tissues occurred
in the albedo before the flavedo, indicating
that the extent of maceration
depends not
only on the quantity
of fungal mycelium
present in the host tissue [Fig. 1 (b)], but also
on a host response. While the levels of galacturonic
acid found in macerated tissues are
similar to those reported
previously
[5, 201, th e subsequent
decrease in the SZ is probably due to fungal utilization
in this region. The lower protein content [Fig. 1 (e)] in
regions where the pH of the water extracts decreased [Fig. 1 (d)] may be partially
due

0. Achilea

132
TABLE

ACC content and conversion of ACC to ethylene by albedo discs, cut from
infected grakefruit

Control
Assayed
site

ACC content
(nmol g- fresh wt)

+70mm
+8mm
-8mm
-7Omm
Albedo discs were
grapefruit.
Five discs
(5 mM) and incubated
was then determined.
180 nmol g- 1 fresh wt.

0.5f0.03
8.8k2.7
18.1 k3.0
32.4k4.2

eta/.

a$erent sites on P. digitatum

ACC

Ethylene production
(nl g- fresh wt h-l)
0.2 + 0.06
2.3kO.8
0.2fO.08
2.OkO.6

Treated

Ethylene production
(90 of control)
994+ 130
142& 31
102+
24
91+
17

excised from the indicated

sites [see Fig. 1 (a)] of P. dig&urn
infected
from each site were treated with either water (control),
or with ACC
at 24 C for 6 h in the dark. The ACC content and ethylene production
The ACC content
of the ACC-treated
discs ranged from 141 to
Each value in the table is a mean of five replicates f SD.

to changes in pH (Table I), but the greater

reductions
in the regions where the fungus
is present in abundance
could be due to degradation
of fruit proteins by the fungus.
Slightly higher amounts of ACC were found in the peel of healthy grapefruit
and in
the HZ of infected grapefruit
than were reported by Yu & Yang [3U] and by Hyodo &
Nishino [16] for healthy Valencia
and Citrus hassaku fruits. However,
these differences
do not explain how infection induced a 23- and log-fold increase in ACC content in the
SZ of the flavedo and the albedo, respectively.
The present study tested whether the
application
of galacturonic
acid [Fig. I(c)], dilute HCI [Fig. l(d)]
or exposure to
ethylene [25j could induce such increases in healthy tissue but none of the treatments
affected ACC content.
Pathogen infection may enhance ACC synthesis either by a wounding
effect caused
by the invading
fungal hyphae on the peel cells or by the action of an inducer produced
by the fungus [Z, 31. Mechanical
and biochemical
stresses are known to initiate ACC
synthesis [9, 26, 301; and Barmore
& Brown
[5j have shown plasmolysed
cells, and
swollen cell walls near to invading
hyphae,
even in the apparently
healthy
zone
( + 8 mm), where fungal hyphae do not penetrate cell walls.
Whatever
the specific signal for the production
of ACC is, its accumulation
in infected peel [Fig. 1 (f)] and as reported
recently, during virus infection
of tobacco leaves
[I,?], suggests that pathogen
infection may affect the formation
of ACC and its subsequent conversion
to ethylene in different ways. Indeed, we showed that in the infected
region of the fruit, the tissue gradually
lost its capacity to convert exogenously
added
ACC to ethylene
(Table 2). Also, in infected fruit, the level of ACC [Fig. 1 (f)] is
probably
not limiting its conversion to ethylene.
Further information
on the processes taking place in infected fruit can be derived
from the data obtained
from the experiments
in which ethylene production
by peel
discs was examined over a period of up to 32 h after excision (Fig. 2). Ethylene production by healthy peel discs was induced by the wounding
stress caused by excision [26J
and attained
maximum
rates about 24 h after excision [Fig. 2(a)]. However,
when

Biochemical-physiological

changes

in infected

133

grapefruit

discs were infected, even by low levels of mycelium,

ethylene production
was suppressed, as shown by the results from albedo disks ( + 8 mm) in Fig. 2 (b) and from flavedo
discs ( -8 mm) 16 h after excision [Fig. 2(c)]. This suppression
of host production
of
ethylene by the developing
fungus probably
results from an impairment
of the conversion of ACC to ethylene which is associated with the cell membrane
[19] since ACC is
still being formed and therefore
accumulates
[Fig. l(f)]. The suppression
does not
occur, due to tissue death because the tissues are still alive as evidenced by the continuing production
of ACC and the high rates of tissue respiration
[27]. The accumulation
of ACC in the infected tissue reported here and by De Laat & Van Loon, who showed a
temporary
accumulation
of ACC in virus-infected
tobacco leaves [12], may be similar
to an earlier observation
on ACC accumulation
in post-climacteric
avocado fruit [14].
In both diseased and overripe
tissues of avocado, the accumulation
of ACC and the
lower rates of ethylene production,
are probably
related to the disintegration
of cell
membranes.
Thus, at advanced stages of infection, where the hosts ethylene production
is suppressed the ethylene, which is still being produced
at high rates [Fig. 1 (g) and
2(d)], is likely to originate
mostly from the fungus [17].
Based on the present findings
the authors suggest the following
hypothesis
to
account for ethylene production
during infection of grapefruit
by P. digitatum. At the
initial phases of infection, for example + 8 mm ahead of the MF, the enhanced production of ethylene originates
from the peel cells and is ACC mediated.
This biosynthetic
pathway is apparently
impaired
by infection since infected tissues from -8 to - 70 mm
behind the MF have a reduced
ability
to convert ACC to ethylene
and so ACC
accumulates.
The increased production
of ethylene at this stage is therefore likely to be
offungal
origin which does not involve ACC as a precursor. This hypothesis is in agreement with earlier reports [IO, 27] which indicate that the pathogen is responsible for the
high rates of ethylene
produced
by infected tissue. At present, the investigation
is
continuing
with the aim of obtaining
direct evidence for the relative contributions
of
host and pathogen
to ethylene production.
This research was supported
in part by a grant
Research & Development
Fund (BARD).

from US-Israel

Binational

Agricultural

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I-carboxylic
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.V&nal
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A. K. &
tobacco leaf discs by cell wall digesting

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LIEBERMAN, M. (1982).
Induction
of ethylene
biosynthesis
in
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588-589.
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Martinus
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