SUMMARY

Rice grain discolouration has a complex etiology and

cannot be diagnosed prior to harvest. In Italy, the disease
was thought to be associated with fungal infections, how-
ever several studies contradicted this hypothesis. We re-
port the distribution of grain discolouration, the most sus-
ceptible Italian rice varieties, and the effects of epidemics
of brown spot, induced by Cochliobolus miyabeanus, and
abiotic factors, such as incidence of grains with open
glumes, chemical treatments, nitrogen fertilization, and
time of sowing, on disease incidence over three years.
Among 39 heavily diseased rice lots, the early cvs Selenio
and Loto were the most susceptible. We did not find any
significant correlation between incidence of grain dis-
colouration and incidence of brown spot. In contrast, in-
cidence of discoloured grains was weakly correlated with
incidence of grains with open glumes (r = 0.521 P =
0.001), while incidence of brown spot was better correlat-
ed with incidence of grains with sealed glumes (r = 0.570,
P < 0.001) than to incidence of grains with open glumes (r
= 0.355, P = 0.026). On susceptible cv. Selenio, percent of
grains with open glumes varied among years, but in-
creased significantly from milk dough stage to fully ripe
maturation of kernels (P < 0.001). The highest percent of
grains with open glumes was found for late-sown rice.
However, late sowing reduced significantly (P 0.001) the
incidence of grain discolouration, whereas chemical and
fungicide treatments had no significant effect. We specu-
late that grain discolouration may have a bacterial rather
than a fungal aetiology.
Key words: Cochliobolus miyabeanus, Oryza sativa;
fungicides, acibenzolar-S-methyl, prohexadione-calci-
um, aetiology.
INTRODUCTION
Rice grain discolouration is a disease of great impor-
tance for its worldwide distribution and the substantial
Corresponding author: P. Cortesi
Fax: +39.02.50316781
E-mail: paolo.cortesi@unimi.it
damage it causes in tropical and sub-tropical countries
(Zeigler and Alvarez, 1987; Duveiller et al., 1988, 1990;
Rott et al., 1989; Cottyn et al., 1996). In Italy, the disease
occurs in all major rice-growing areas with incidence
ranging from 1% to 30%; medium- and short-grain rice
varieties are more susceptible than the long-grain ones
(Pizzatti et al., 2004; Pizzatti, 2005). On milled rice, dis-
coloured grains show brownish-black irregular lesions,
chalkiness and shrivelling, and their incidence in milled
rice lots cannot exceed 1.5%. To meet this threshold,
mills sustain additional costs to discard discoloured
grains, therefore they reduce the price paid to the farmers
by about 2.5 /ton for each 1% of discoloured grains
above the threshold (Cortesi, unpublished information).
Grain discolouration has a complex etiology and can-
not be diagnosed prior to harvest based on symptoms. In
tropical and sub-tropical countries it was associated with
sheath rot, blast and brown spot epidemics caused by the
pathogenic fungi Sarocladium oryzae, Magnaporthe oryzae
and Cochliobolus miyabeanus, respectively, as well as with
sheath brown rot, grain rot and bacterial brown stripe in-
duced by the pathogenic bacteria Burkholderia glumae,
Pseudomonas fuscovaginae and Acidovorax avenae subsp.
avenae, respectively (Ou, 1985; Zeigler and Alvarez,
1987; Zeigler et al., 1987; Webster and Gunnel, 1992;
Cottyn et al., 1996). In Italy, the rice grain microflora is
composed of both pathogenic and saprophytic fungi,
such as C. miyabeanus, M. oryzae, Alternaria spp., Epicoc-
cum spp., Penicillium spp., Nigrospora spp. and Phoma
spp. (Caufin and Moletti, 1988; Picco et al., 2002), and
unidentified bacteria (Baldacci and Corbetta, 1964).
Cochliobolus miyabeanus was the only fungal pathogen
isolated from discoloured grains with appreciable fre-
quency (Cofelice et al., 2002).
The presence of C. miyabeanus on discoloured grains
suggested that it had a primary role in grain discoloura-
tion aetiology (Fazli and Schroeder, 1966; Haegi et al.,
2002). However, several evidences contradict this hypoth-
esis. First, in Italy and Texas (USA), fungicide treatments
controlled brown spot epidemics, but they were ineffec-
tive against grain discolouration (Marchetti and Petersen,
1984; Cortesi and Giuditta, 2003) and, unexpectedly, in-
cidence of discoloured grains was higher for rice from
treated than from the untreated plots (Moletti et al.,
Journal of Plant Pathology (2008), 90 (2), 197-209 Edizioni ETS Pisa, 2008 197
EFFECT OF CHEMICALS, NITROGEN, TIME OF SOWING AND PANICLE
BROWN SPOT EPIDEMICS ON RICE GRAIN DISCOLOURATION IN ITALY
C. Pizzatti and P. Cortesi
Istituto di Patologia Vegetale, Universit degli Studi, Via Celoria 2, 20133 Milano, Italy
006_JPP_77RP_197 21-07-2008 10:24 Pagina 197
1996). Second, brown spot incidence on spikelets was
rarely correlated with incidence of discoloured grains
and, furthermore, a poor correlation was found between
the isolation frequency of C. miyabeanus and grain dis-
colouration (Marchetti and Petersen, 1984; Cofelice et al.,
2002). These results would support the hypothesis of a
secondary role of C. miyabeanus on grain discolouration.
The infestations of the stink bug Oebalus pugnax and
nitrogen fertilization may enhance grain discolouration.
Severe infestations of the stink bug and the bug Lepto-
coria oratorius were highly correlated with the incidence
of discoloured grains. However, O. pugnax was not re-
sponsible for grain discolouration, although it may have
favoured the spread of pathogens through the feeding
sheaths (Marchetti and Petersen, 1984). Nitrogen may
act in the same way, favouring the formation of early en-
try sites subsequently infected by microbes. In fact,
grains from paddies fertilized with high amounts of ni-
trogen showed unsealed lemmas and paleas, and dis-
coloured pericarps were visible through the openings,
suggesting the hypothesis that the disease was correlat-
ed to the percentage of grains with unsealed glumes
(Moletti et al., 1996). Finally, the role of the leaf bug
Trigonotylus caelestialium as a causal agent of the dis-
ease was recently proposed, based on greenhouse ex-
periments, but no data on the population size were re-
ported (Giudici and Villa, 2006). Since the incidence of
grain discolouration in the USA and in the Philippines
was positively correlated only with severe infestations of
rice bugs (Marchetti and Petersen, 1984; Jahn et al.,
2004), absence of biological and population size data
for T. caelestialium leaves an open question about its
role in the aetiology of grain discolouration.
In Italy, to our knowledge, the aetiology of grain dis-
colouration is still unknown, because of the lack of ex-
perimental results supporting the role of C. miyabeanus,
nitrogen fertilization, open glumes or insects infestation
(Moletti et al., 1996; Cofelice et al., 2002; Haegi et al.,
2002; Giudici and Villa, 2006).
To clarify the uncertain and complex interactions
between grain discolouration, brown spot and some
abiotic factors, we conducted this research with the fol-
lowing objectives: (i) to identify the Italian rice varieties
most susceptible to this disorder; (ii) to investigate the
correlations between incidence of discoloured grains,
brown spot and grains with open glumes, and (iii) to
assess if chemical treatments, date of sowing and nitro-
gen fertilization may affect the incidence of rice grain
discolouration.
MATERIALS AND METHODS
Incidence of grain discolouration and rice variety
susceptibility. A survey was carried out in collaboration
with the Rice Research Centre, Castello dAgogna
(Pavia, northern Italy) of the Ente Nazionale Risi
(ENR). In 2001, technicians at the ENR Provincial of-
fices retained 1 kg of rough rice for each rice lot sample
exceeding 4% and 2.75% of discoloured grains for
short- and medium-grain, and long-grain varieties, re-
spectively. We chose these thresholds because they can-
not be overcome for rough rice to be sold on the inter-
national market. From each sample, 100 g of rough rice
was milled and the amount of whole kernel weighted
(g). Whole kernels were visually observed and dis-
coloured grains were separated and weighted (g). For
milled rice, the percent of discoloured grains was calcu-
lated as discoloured grains/whole kernels (w/w). Dis-
ease distribution was determined as frequencies of dis-
eased samples per area of origin and per variety. Rice
variety susceptibility was determined based on average
incidence of discoloured grains.
All ENR samples were delivered to the Institute of
Plant Pathology, University of Milan, for further analy-
ses. To determine the level of correlation between inci-
dence of brown spot and grain discolouration, between
incidence of brown spot and grains with open glumes,
and between incidence of grain discolouration and open
glumes two sub-samples of 100 grains of rough rice
were randomly taken from each diseased sample. One
was used to determine incidence of grains with symp-
toms of brown spot, by visually checking the glumes.
The other was used to determine the incidence of grains
with open glumes by forcing lemma and palea to open
by applying a small pressure at the tips of the grain with
a forceps. Then, grains from the two sub-samples,
named grains with open glumes and grains with
sealed glumes, were hand-hulled, and the brown rice
was visually checked to separate discoloured grains. In-
cidence of grain discolouration for each sub-sample was
calculated as percent of discoloured grains.
Field experiments. Field experiments were designed
to test the role of chemical treatments, date of sowing
and nitrogen fertilization on the incidence and severity
of brown spot and on the incidence of grain discoloura-
tion, both on grains with open glumes and sealed
glumes. The experiment was designed as a three-way
factorial randomized complete block split-plot design
with four replications in 1999 and 2000, and with three
replications in 2001. Each simple plot was 21 m
2
. Field
trials were carried out at Rosasco (Pavia province, Italy)
using Oriza sativa subsp. japonica, early cv. Selenio,
known to be susceptible to grain discolouration (Pizzatti
et al., 2004). The chemicals used were two fungicides,
azoxystrobin and tricyclazole, the Systemic Acquired Re-
sistance (SAR) activator acibenzolar-S-methyl, and the
growth regulator prohexadione-calcium, known for be-
ing effective against some bacterial pathogens on specific
host-pathogen combinations (Friedrich et al., 1996;
Lawton et al., 1996; Ruess et al., 1996). Acibenzolar-S-
198 Rice grain discolouration Journal of Plant Pathology (2008), 90 (2), 197-209
006_JPP_77RP_197 21-07-2008 10:24 Pagina 198
methyl was also tank mixed with tricyclazole and with
prohexadione-calcium. Chemicals were applied as single
or double treatments between the end of tillering and
heading (Table 1), spraying 500 l/ha of an aqueous solu-
tion with the motorised backpack sprayer Fox F320 and
an hand-held 1.5 m boom, operating at 500 Kpa. Each
chemical-treated plot was subdivided for the two sub-
plot factors: time of sowing (early and late), and nitrogen
fertilization (high and low). For early sowing, rice was
seeded by mid-April (between 9 and 17 April), whereas
for late sowing it was seeded by mid-May (between 6
and 10 May). The two levels of nitrogen fertilization dif-
fered by 30 kg/ha and the difference was obtained with
the top-dress fertilization, applying 50 and 80 kg/ha of
N as urea, at late tillering (panicle 1-2 mm in length) to
plots fertilized with low and high level of N, respectively.
Panicle brown spot incidence (as percent of infected
panicle) and severity (as percent infected panicle area)
were assessed at harvest by visually checking 50 ran-
domly chosen panicles in each simple plot, and the effi-
cacy of each chemical was calculated by comparison
with untreated check plots (Cortesi and Giuditta, 2003).
In 1999 and 2000, incidence of grains with open
glumes was monitored at three phenological stages of
fruit development: early milk (mid- and end-August for
early and late sowing, respectively), hard dough (early-
and mid-September for early and late sowing, respective-
ly) and at harvest (mid- and end-September-early-Octo-
ber for early and late sowing, respectively), whereas in
2001 it was assessed at harvest (mid-September and ear-
ly-October for early and late sowing, respectively) (Lan-
cashire et al., 1991). In 1999 and 2000, ten panicles were
randomly harvested from each simple plot, allowed to
dry in the shade, and hand-threshed. In 2001, a sample
of about 0.5 kg of rough rice was taken from each simple
plot at harvest. Incidence of grains with glumes open or
sealed was determined as described above.
Incidence of grain discolouration was assessed for
the two sub-samples, open glumes and sealed
glumes in 2000 and 2001. Following hulling, each sam-
ple of 25 grains was visually checked to separate dis-
coloured grains and percent of discoloured grains was
calculated.
Rice was harvested at maturity with an Iseki har-
vester. A sample of 100 g of air-dried rough rice from
each simple plot was used to determine the milling yield
and the percent of discoloured grains on milled rice as
described above.
Data analysis. The data obtained from ENR samples
were submitted to correlation analysis using the MSTAT-
C statistical package (Nissen, 1983). We tested for signifi-
cant correlations, P 0.05, between incidence of brown
spot and grain discolouration, between incidence of
brown spot and grains with glumes open, and between
incidence of grain discolouration and grains with glumes
open. The data obtained from field experiments were
submitted to analysis of variance (ANOVA) with a gener-
al linear model for split-plot experimental design, and
Duncans multiple range tests using MSTAT-C statistical
package. Significance of interactions was evaluated first
at P 0.20. Data expressed as percentage were arcsin-
square-root-transformed to stabilize variances.
RESULTS
Rice variety susceptibility and factors correlated
with incidence of grain discolouration. The survey
showed the 39 diseased samples were mostly from Pied-
mont (north west Italy) and included 13 varieties: 5
short-grain, 7 medium-grain and 1 long-grain (Fig. 1).
Incidence of grain discolouration of the most suscepti-
Journal of Plant Pathology (2008), 90 (2), 197-209 Pizzatti and Cortesi 199
Fig. 1. Distribution of discoloured rice samples (N = 39)
among provinces (A) in the Piedmont region () and in the
Lombardy region ( ), and (B) among varieties grouped by
grain type.
006_JPP_77RP_197 21-07-2008 10:24 Pagina 199
ble short-grain cv. Selenio and medium-grain cv. Loto
was 13.7% (s.e. = 1.6; N = 18) and 14.6% (s.e. = 1.8; N
= 7), respectively. In 2001, cvs Selenio and Loto were
grown on 9% and 12% of the rice area, respectively.
Incidence of brown spot was not correlated with in-
cidence of discoloured grains and with incidence of
grains with open glumes as well. In contrast, incidence
of discoloured grains was slightly but significantly corre-
lated with incidence of grains with open glumes for
short-grain and all varieties pooled, but not for medi-
um-grain varieties. Finally, we showed that incidence of
brown spot was better correlated with incidence of
grains with sealed glumes, than with incidence of grains
with open glumes (Table 2).
Dynamics of grains with open glumes. On cv. Sele-
nio, percent of grains with open glumes increased sig-
nificantly (P < 0.001) from milk dough stage to harvest
of kernels, from 2.9% to 19.1% in 1999, and from
46.7% to 54.8%, in 2000. In 2001, at harvest it was
36,6%. A significant increase (P < 0.001) occurred at
high level of nitrogen, from 8.9% to 11.8% in 1999 and
from 47.8% to 60.3% in 2000. The date of sowing in-
fluenced significantly (P < 0.001) the incidence of grains
with open glumes. However, in 2000, it increased from
45.8% to 62.4% delaying the sowing, whereas in 2001 it
decreased from 41.1% to 32.1%. In 2000, the interac-
tion between nitrogen fertilization and date of sowing
was significant (P < 0.001) and the highest percent of
grains with open glumes was 71.3%, for plot with high
nitrogen and late sowing. The effect of the chemicals
was significant (P < 0.003) in 2000 only, with the highest
percentage of grains with open glumes, 60.1%, found
on plots treated with azoxystrobin (Tables 3 and 4).
Incidence of grain discolouration for rice with open
and sealed glumes. Average incidence of discoloured
grain was 2.9% in 2000 and 6.3% in 2001. In 2000, dis-
ease incidence increased from hard dough phenological
stage to harvest, and in both years it was significantly
higher (P 0.001) on grains with open glumes, 5% and
9.8%, than on grains with sealed glumes, 0.8% and
2.9%. In both years, neither the chemicals nor the ni-
trogen fertilization had a significant effect on the disease
incidence. On the contrary, the date of sowing had a sig-
nificant effect (P 0.001) and higher disease incidence
was found on early sown rice, 4.6% and 9%, than on
late sown rice, 1.2% and 3.7%, in 2000 and 2001 re-
spectively. The interaction between the date of sowing
and the glumes type was significant only in 2000, and
the highest disease incidence, 8.2%, was found for early
sown rice with open glumes, while the interaction of the
two factors with the nitrogen fertilization did not signif-
icantly change disease incidence. The interaction be-
tween the chemicals and the date of sowing was signifi-
cant only in 2001 (P < 0.08), and the disease increased
200 Rice grain discolouration Journal of Plant Pathology (2008), 90 (2), 197-209
T
a
b
l
e

1
.
C
h
e
m
i
c
a
l
s
,

a
p
p
l
i
c
a
t
i
o
n

r
a
t
e
,

s
p
r
a
y

s
c
h
e
d
u
l
e
,

g
r
o
w
t
h

s
t
a
g
e

a
n
d

d
a
t
e

o
f

t
r
e
a
t
m
e
n
t

o
n

r
i
c
e

c
v
.

S
e
l
e
n
i
o

a
t

R
o
s
a
s
c
o

(
P
a
v
i
a
)
,

I
t
a
l
y
.
a

A
p
p
l
i
c
a
t
i
o
n

r
a
t
e

i
n

1
9
9
9
;

b

A
p
p
l
i
c
a
t
i
o
n

r
a
t
e

i
n

2
0
0
0

a
n
d

2
0
0
1
S
p
r
a
y

s
c
h
e
d
u
l
e

a
n
d

d
a
t
e

o
f

a
p
p
l
i
c
a
t
i
o
n
1
9
9
9
2
0
0
0
2
0
0
1
C
h
e
m
i
c
a
l
s
A
p
p
l
i
c
a
t
i
o
n
r
a
t
e
(
m
l
,

g

a
.
i
.
/
h
a
)
G
r
o
w
t
h
s
t
a
g
e
E
a
r
l
y
s
o
w
i
n
g
L
a
t
e
s
o
w
i
n
g
G
r
o
w
t
h
s
t
a
g
e
E
a
r
l
y
s
o
w
i
n
g
L
a
t
e
s
o
w
i
n
g
G
r
o
w
t
h
s
t
a
g
e
E
a
r
l
y
s
o
w
i
n
g
L
a
t
e
s
o
w
i
n
g
T
r
i
c
y
c
l
a
z
o
l
e

(
B
e
a
m

W
P

-

D
o
w

A
g
r
o
s
c
i
e
n
c
e
s
)
4
5
0
H
e
a
d
i
n
g
2
7

J
u
l
y
6

A
u
g
u
s
t
H
e
a
d
i
n
g
2
9

J
u
l
y
1
2

A
u
g
u
s
t
H
e
a
d
i
n
g
3
1

J
u
l
y
1
4

A
u
g
u
s
t
A
z
o
x
y
s
t
r
o
b
i
n

(
A
m
i
s
t
a
r

S
C

-

S
y
n
g
e
n
t
a
)
3
1
2
.
5
a

a
n
d
2
5
0
b
H
e
a
d
i
n
g
2
7

J
u
l
y
6

A
u
g
u
s
t
H
e
a
d
i
n
g
2
9

J
u
l
y
1
2

A
u
g
u
s
t
H
e
a
d
i
n
g
3
1
J
u
l
y
1
4

A
u
g
u
s
t
A
c
i
b
e
n
z
o
l
a
r
-
S
-
m
e
t
h
y
l

(
B
i
o
n


W
G

-

S
y
n
g
e
n
t
a
)
7
0
H
e
a
d
i
n
g
2
7

J
u
l
y
6

A
u
g
u
s
t
H
e
a
d
i
n
g
2
9

J
u
l
y
1
2

A
u
g
u
s
t
-
-
-
A
c
i
b
e
n
z
o
l
a
r
-
S
-
m
e
t
h
y
l
7
0
E
n
d

o
f

t
i
l
l
e
r
i
n
g
+

h
e
a
d
i
n
g
7

+

2
7

J
u
l
y
7

J
u
l
y

+

6
A
u
g
u
s
t
H
e
a
d
i
n
g

+

2
w
k
s

a
f
t
e
r
2
9

J
u
l
y

+

1
2
A
u
g
u
s
t
1
2

+

2
4
A
u
g
u
s
t
-
-
-
T
r
i
c
y
c
l
a
z
o
l
e

+

a
c
i
b
e
n
z
o
l
a
r
-
S
-
m
e
t
h
y
l
4
5
0

+

7
0
H
e
a
d
i
n
g
2
7

J
u
l
y
6

A
u
g
u
s
t
-
-
-
-
-
-
A
z
o
x
y
s
t
r
o
b
i
n

+

a
c
i
b
e
n
z
o
l
a
r
-
S
-
m
e
t
h
y
l
3
1
2
.
5

+

7
0
H
e
a
d
i
n
g
2
7

J
u
l
y
6

A
u
g
u
s
t
-
-
-
-
-
-
P
r
o
h
e
x
a
d
i
o
n
e
-
C
a

(
R
e
g
a
l
i
s

-

B
a
s
f
)
2
0
H
e
a
d
i
n
g
2
7

J
u
l
y
6

A
u
g
u
s
t
H
e
a
d
i
n
g

+

2
w
k
s

a
f
t
e
r
2
9

J
u
l
y

+
1
2
A
u
g
u
s
t
1
2

+

2
4
A
u
g
u
s
t
-
-
-
P
r
o
h
e
x
a
d
i
o
n
e
-
C
a

+

a
c
i
b
e
n
z
o
l
a
r
-
S

m
e
t
h
y
l
2
0

+

7
0
H
e
a
d
i
n
g
2
7

J
u
l
y
6

A
u
g
u
s
t
-
-
-
-
-
-
006_JPP_77RP_197 21-07-2008 10:24 Pagina 200
significantly, from 2.3% to 5.7%, on rice of the late
sowing plots treated with tricyclazole (Tables 5 and 6).
Panicle brown spot and grain discolouration epi-
demics and milling yield. Symptoms of brown spot were
found on 21.3%, 90.9% and 100% of untreated panicle
in 1999-2001, respectively, and the corresponding severi-
ty was 11.8%, 62.5% and 21.5%. Although the chemi-
cals had a significant effect on the disease in 1999 and in
2000 (P 0.02 for incidence and severity), only azoxys-
trobin in 2000 determined a significant reduction of
brown spot incidence and severity. In 2000 and 2001,
years of more severe epidemics, low nitrogen input sig-
nificantly increased panicle brown spot incidence in
2000, and severity in both years (P = 0.045 for incidence
in 2000; P = 0.07 and P < 0.001 for severity in 1999 and
2000, respectively). Less consistent was the effect of the
date of sowing. On early sown rice, the most severe epi-
demic occurred in 1999 (P < 0.001 for incidence and
severity), whereas disease severity was lower in 2000 (P =
0.053). The interaction between nitrogen fertilization
and date of sowing was significant, but the effect was not
consistent. In 1999, lower incidence of brown spot was
found on late sown rice treated with high input of nitro-
gen, whereas in 2001 was the opposite (Table 7 and 8).
Average incidence of grain discolouration, was 0.8%
in 1999, 1.9% in 2000 and 3.1% in 2001. High input of
nitrogen significantly (P < 0.001) increased the disease
from 1.6% to 2.3% in 2000, whereas the delay of date
of sowing reduced significantly (P < 0.001) discoloured
grains: from 3% to 0.8% in 2000 and from 4% to 1.9%
in 2001 (Table 7 and 8).
Milling yield was not influenced by chemical treat-
ments, whereas high input of nitrogen in 2000 and in
2001 (P = 0.005 and P = 0.01, respectively), and late
sowing in all three years (P 0.001), had a significant
positive effect (Table 7 and 8).
DISCUSSION
In Italy, grain discolouration of rice adds to other grain
defects, such as greening, shrivelling, chalkiness etc., low-
ering quality and market value. The disease has a contro-
versial etiology and is a common problem with some rice
varieties (Moletti et al., 1996; Infantino et al., 1997; Cofe-
lice et al., 2002; Cortesi and Giuditta, 2003; Pizzatti,
2005). Our survey showed that grain discolouration is
widespread, but it occurs more frequently on short-grain
than on medium-grain varieties, whereas long-grain vari-
eties, grown on roughly 60% of rice area, are rarely af-
fected. Early varieties, such as the short-grain cv. Selenio
and the medium-grain cv. Loto, are the most susceptible,
however these varieties are more often diseased in No-
vara and Vercelli provinces than in Pavia province, which
alone has the largest area grown with cvs. Loto and Sele-
Journal of Plant Pathology (2008), 90 (2), 197-209 Pizzatti and Cortesi 201
T
a
b
l
e

2
.
C
o
r
r
e
l
a
t
i
o
n

b
e
t
w
e
e
n

i
n
c
i
d
e
n
c
e

o
f

b
r
o
w
n

s
p
o
t

a
n
d

i
n
c
i
d
e
n
c
e

o
f

g
r
a
i
n

d
i
s
c
o
l
o
u
r
a
t
i
o
n
,

g
r
a
i
n
s

w
i
t
h

o
p
e
n

g
l
u
m
e
s

a
n
d

s
e
a
l
e
d

g
l
u
m
e
s

o
n

r
i
c
e

s
a
m
p
l
e
s

o
f

s
h
o
r
t
-

a
n
d

m
e
d
i
u
m
-
g
r
a
i
n

v
a
r
i
e
t
i
e
s

c
o
l
l
e
c
t
e
d

i
n

P
i
e
d
m
o
n
t

a
n
d

i
n

L
o
m
b
a
r
d
y

i
n

2
0
0
1
.
a


A
l
l

s
a
m
p
l
e
s

p
o
o
l
e
d
.

b

S
h
o
r
t
-
g
r
a
i
n

r
i
c
e

v
a
r
i
e
t
i
e
s
.

c

M
e
d
i
u
m
-
g
r
a
i
n

r
i
c
e

v
a
r
i
e
t
i
e
s
.
I
n
c
i
d
e
n
c
e

o
f

g
r
a
i
n

d
i
s
c
o
l
o
u
r
a
t
i
o
n

(
%
)
I
n
c
i
d
e
n
c
e

o
f

g
r
a
i
n
s

w
i
t
h

o
p
e
n

g
l
u
m
e
s

(
%
)
I
n
c
i
d
e
n
c
e

o
f

g
r
a
i
n
s

w
i
t
h

s
e
a
l
e
d

g
l
u
m
e
s

(
%
)
F
a
c
t
o
r
N
r
P
r
P
r
P
3
9
a
-
0
.
1
0
6
0
.
4
2
5
-
0
.
2
9
7
0
.
2
0
6
2
4
b
-
0
.
1
2
0
0
.
5
7
5
-
0
.
2
1
7
0
.
3
0
8
I
n
c
i
d
e
n
c
e

o
f

b
r
o
w
n

s
p
o
t

(
%
)
1
4
c
-
0
.
1
1
6
0
.
6
9
2
-
0
.
4
2
2
0
.
1
3
1
3
9
0
.
5
2
1
0
.
0
0
1
2
4
-
0
.
6
0
6
0
.
0
0
2
I
n
c
i
d
e
n
c
e

o
f

g
r
a
i
n
s

w
i
t
h

o
p
e
n

g
l
u
m
e
s

(
%
)
1
4
0
.
4
0
4
0
.
1
5
0
3
9
0
.
3
5
5
0
.
0
2
6
2
4
0
.
4
2
6
0
.
0
3
8
I
n
c
i
d
e
n
c
e

o
f

b
r
o
w
n

s
p
o
t

o
n

g
r
a
i
n
s

w
i
t
h

o
p
e
n
g
l
u
m
e
s

(
%
)
1
4
0
.
1
2
1
0
.
6
7
9
3
9
0
.
5
7
0
<
0
.
0
0
1
2
4
0
.
5
7
3
0
.
0
0
3
I
n
c
i
d
e
n
c
e

o
f

b
r
o
w
n

s
p
o
t

o
n

g
r
a
i
n
s

w
i
t
h

s
e
a
l
e
d
g
l
u
m
e
s

(
%
)
1
4
0
.
8
1
5
<
0
.
0
0
1
006_JPP_77RP_197 21-07-2008 10:24 Pagina 201
nio (www.enterisi.it). Therefore, the spatial distribution
and incidence of grain discolouration are not strictly re-
lated with varietal susceptibility, but other factors such as
crop cycle, environment or spatial distribution of the
pathogen/s may modulate disease epidemics.
Cochliobolus miyabeanus was suggested as the most
important causal agent of grain discolouration because
it produces toxins (Infantino et al., 1997; Cofelice et al.,
2002). However, these toxins cause discolouration ex-
clusively following early infections and partial removal
of the glumes (Haegi et al., 2002). This finding agrees
with early observation by Moletti et al. (1996), who
found discolouration of grains localized underneath
open glumes, entry sites for several species of fungi, C.
miyabeanus included. In our study, we did not find evi-
dence supporting the C. miyabeanus aetiology of grain
discolouration. First, we showed with several varieties,
including the susceptible cv. Selenio, that incidence of
grains with symptoms of brown spot was not correlated
with either incidence of discoloured grains (r = -0.106,
P = 0.425) and incidence of grains with open glumes (r
= -0.207, P = 0.206). Second, incidence of grains with
symptoms of brown spot was more correlated with inci-
dence of grains with sealed glumes (r = 0.570, P <
0.001), than with incidence of grains with open glumes
(r = 0.355, P = 0.026). Third, incidence of discoloured
grains was significant but slightly correlated with per-
cent of grains with open glumes, even for short-grain
varieties (r = 0.606, P = 0.002). We conclude that grain
discolouration cannot be attributed to epidemics of C.
miyabeanus, but it may have a different and complex ae-
tiology, hypothesis supported by the lack of fungicides
efficacy against the disease (see also Moletti et al., 1996;
Cortesi and Giuditta, 2003)
In our field experiment, we tested, on the susceptible
cv. Selenio, whether incidence of grain discolouration
and frequency of grains with open glumes were influ-
enced by epidemics of panicle brown spot, or by abiotic
factors such as the date of sowing, the rate of nitrogen
and chemicals and fungicide treatments. We provided
evidence that panicle brown spot epidemics, incidence
of grain discolouration and frequency of grains with
open glumes always increased during rice maturation,
but the final values of the three parameters varied inde-
pendently over the years. The highest incidence of grain
discolouration occurred in 2001, but it was not associat-
ed to the highest frequency of grains with open glumes
nor with the highest severity of brown spot occurred in
2000. The delay of the date of sowing, from mid-April
to mid-May was the most important factor for reducing
202 Rice grain discolouration Journal of Plant Pathology (2008), 90 (2), 197-209
Table 3. Effect of chemicals, nitrogen fertilization and date of sowing on incidence of grains with open glumes of rice cv. Selenio,
at three phenological stages of fruit development, at Rosasco (Pavia), Italy.
Grains with open glumes (%) Factor
1999 2000 2001
Untreated control 10.14
b
n.s.
c
52.75 b
d
32.0 n.s.
Tricyclazole 9.64 54.52 b 37.67
Azoxystrobin 10.11 60.12 a 40.17
Acibenzolar-S-methyl 9.53 53.67 b -
Acibenzolar-S-methyl (2 treatments) 9.47 51.58 b -
Tricyclazole + Acibenzolar-S-methyl 9.09 - -
Azoxystrobin + Acibenzolar-S-methyl 13.86 - -
Prohexadione-Ca 12.06 55.12 b -
Prohexadione-Ca (2 treatments) - 50.71 b -
Chemicals
a
Prohexadione-Ca + Acibenzolar-S-methyl 9.31 - -
Low 8.94 a 47.85 a 35.89 n.s. N-fertilization
High 11.76 b 60.29 b 37.33
Early (Mid-April) 10.25 n.s. 45.77 a 41.11 a Date of sowing
Late (Mid-May) 10.46 62.36 b 32.11 b
1
st
milk dough (End-August) 2.86 a 46.65 a -
2
nd
hard dough (Mid-September) 9.11 b 60.79 c
Date of sampling
3
rd
harvest (Mid-October) 19.09 c 54.77 b
a
See table 1 for details.
b
Mean value.
c
Not significant.
d
Duncans multiple range test P = 0.05. Means with the same letter are not
significantly different.
006_JPP_77RP_197 21-07-2008 10:24 Pagina 202
J
o
u
r
n
a
l

o
f

P
l
a
n
t

P
a
t
h
o
l
o
g
y

(
2
0
0
8
)
,

9
0
(
2
)
,

1
9
7
-
2
0
9
P
i
z
z
a
t
t
i

a
n
d

C
o
r
t
e
s
i
2
0
3
Table 4. ANOVA to test the effects of chemicals, nitrogen fertilization, date of sowing and date of sampling on incidence of grains with open glumes of rice cv. Selenio at Rosas-
co (Pavia), Italy.
1999 2000 2001
Source
df MS
a
F P df MS F P df MS F P
Replication 2 0.031 2.879 0.086 3 0.319 37.052 < 0.001 2 0.047 2.658 0.184
Chemicals (A) 8 0.026 2.418 0.063 6 0.058 6.723 < 0.001 2 0.025 1.407 n.s.
Error 16 0.011 18 0.009 4 0.018
N fertilization (B) 1 0.168 9.471 0.007 1 1.574 170.519 < 0.001 1 0.001 0.120 n.s.
A x B 8 0.009 0.536 n.s.
b
6 0.006 0.699 n.s. 2 0.007 0.774 n.s.
Error 18 0.018 21 0.009 6
Date of sowing (C) 1 0.016 1.083 n.s. 1 2.683 143.974 < 0.001 1 0.083 52.695 < 0.001
A x C 8 0.004 0.293 n.s. 6 0.011 0.601 n.s. 2 0.010 6.465 0.012
B x C 1 0.003 0.177 n.s. 1 0.289 15.517 < 0.001 1 0.002 1.056 n.s.
A x B x C 8 0.013 0.866 n.s. 6 0.027 1.451 0.197 2 0.002 1.015 n.s.
Error - - - - - - - - 12 0.002
Sampling date (D) 2 2.688 179.213 < 0.001 2 0.714 38.296 < 0.001
A x D 16 0.017 1.164 n.s. 12 0.030 1.583 0.098
B x D 2 0.008 0.564 n.s. 2 0.038 2.053 0.131
A x B x D 16 0.012 0.778 n.s. 12 0.023 1.209 n.s.
C x D 2 0.125 8.343 < 0.001 2 1.377 73.895 < 0.001
A x C x D 16 0.013 0.864 n.s. 12 0.014 0.754 n.s.
B x C x D 2 0.008 0.506 n.s. 2 0.323 17.322 < 0.001
A x B x C x D 16 0.011 0.728 n.s. 12 0.010 0.520 n.s.
Error 180 0.015 210 0.019
Total 323 335 35
a
Mean square.
b
P values > 0.2 were indicated as not significant.
0
0
6
_
J
P
P
_
7
7
R
P
_
1
9
7


2
1
-
0
7
-
2
0
0
8


1
0
:
2
4


P
a
g
i
n
a

2
0
3
incidence of grain discolouration, despite of significant
increase of incidence of grains with open glumes for the
late sowing. On the contrary, nitrogen fertilization and
chemical treatments did not have any significant effects,
with the exception of azoxystrobin, which increased in-
cidence of grains with open glumes. Propiconazole and
iprodione, and high input of nitrogen significantly in-
creased incidence of discoloured grains on the short-
grain cv. Elio (Moletti et al., 1996), results not con-
firmed in our study for cv. Selenio.
Altogether our results indicate that in Italy grain dis-
colouration is restricted to early varieties with short- or
medium-grain, and it occurs more frequently in the
provinces of Piedmont than Lombardy. After a thor-
ough testing of several hypotheses, we conclude that in-
cidence of grain discolouration is not correlated with in-
fections of C. miyabeanus to panicles, but is weakly cor-
related with percentage of grains with open glumes. Sig-
nificant less disease can be obtained delaying the date of
sowing, although late sowing determines the greatest in-
crease of grains with open glumes. Therefore, openings
between glumes could be entry sites for pathogens caus-
ing outbreaks only when favourable environmental con-
ditions meet susceptible plant growth stages.
In tropical and sub-tropical countries the seed-borne
bacteria Burkholderia glumae, Pseudomonas fuscovaginae
and Acidovorax avenae subsp. avenae are reported as
causal agents of grain discolouration on symptomless
panicles (Zeigler and Alvarez, 1990; Cottyn et al., 1996).
We recently reported the presence of A. avenae subsp.
avenae in Italy (Cortesi et al., 2005), and we found the
bacterium associated with sterile panicles and dis-
coloured grains (Cortesi et al., 2008). In this study, how-
ever, we failed to control grain discolouration with
acibezolar-S-methyl and prohexadione-calcium. This re-
sult will not rule out the bacterial aetiology, since these
compounds are effective only against some bacterial
pathogens (Friedrich et al., 1996; Lawton et al., 1996;
Ruess et al., 1996). Investigating the bacterial aetiology of
grain discolouration will be the next step of our research.
ACKNOWLEDGEMENTS
We thank Alessandro Bermano for allowing us to con-
duct this study in his farm, and for technical assistance.
We also thank researchers and technicians of the Ente
Nazionale Risi, Centro Ricerce sul Riso, Castello dA-
gogna (PV), Italy, for technical assistance. This research
was funded in part by Ente Nazionale Risi, Milan, Italy.
204 Rice grain discolouration Journal of Plant Pathology (2008), 90 (2), 197-209
Table 5. Effect of chemicals, nitrogen fertilization and date of sowing on incidence of grain discolouration for rice cv. Selenio with
open and sealed glumes at Rosasco (Pavia), Italy.
Grain discolouration (%)
2000 2001
Factor
Hard dough Harvest Harvest
Chemicals
a
Untreated control 1.75
b
n.s.
c
4.06 n.s. 5.67 n.s.
Tricyclazole
0.75 2.13 6.83
Azoxystrobin 0.50 2.00 6.50
Acibenzolar-S-methyl 0.88 3.00 -
Acibenzolar-S-methyl (2 treatments) 0.50 3.13 -
Tricyclazole + Acibenzolar-S-methyl - - -
Azoxystrobin + Acibenzolar-S-methyl - - -
Prohexadione-Ca 1.00 2.88 -
Prohexadione-Ca (2 treatments) 0.63 3.13 -
Prohexadione-Ca + Acibenzolar-S-methyl - - -
N-fertilization Low 0.79 n.s. 2.71 n.s. 6.67 n.s.
High 0.93 3.09 6.00
Date of sowing Early (Mid-April) 1.32 a
d
4.63 a 9.00 a
Late (Mid-May) 0.39 b 1.18 b 3.67 b
Glumes type Open glumes 1.32 a 5.02 a 9.78 a
Sealed glumes 0.39 b 0.79 b 2.89 b
a
See table 1 for details.
b
Mean value.
c
Not significant.
d
Duncans multiple range test P = 0.05. Mean with the same letter are not
significantly different.
006_JPP_77RP_197 21-07-2008 10:24 Pagina 204
J
o
u
r
n
a
l

o
f

P
l
a
n
t

P
a
t
h
o
l
o
g
y

(
2
0
0
8
)
,

9
0
(
2
)
,

1
9
7
-
2
0
9
P
i
z
z
a
t
t
i

a
n
d

C
o
r
t
e
s
i
2
0
5
Table 6. ANOVA to test the effects of chemicals, nitrogen fertilization and date of sowing on incidence of grain discolouration for rice cv. Selenio with open and sealed glumes at
Rosasco (Pavia), Italy.
2000 2001
Hard dough Harvest Harvest
Source
df MS
a
F P df MS F P df MS F P
Replication 3 0.019 2.926 0.062 3 0.010 0.744 n.s.
b
2 0.004 0.102 n.s.
Chemicals (A) 6 0.012 1.755 0.166 6 0.008 0.585 n.s. 2 0.016 0.448 n.s.
Error 18 0.007 18 0.014 4 0.035
N fertilization (B) 1 0.002 0.238 n.s.
b
1 0.001 0.097 n.s. 1 0.003 0.122 n.s.
A x B 6 0.004 0.421 n.s. 6 0.003 0.233 n.s. 2 0.005 0.174 n.s.
Error 21 0.010 21 0.014 6 0.028
Date of sowing (C) 1 0.066 10.910 0.001 1 0.490 36.370 < 0.001 1 0.283 29.002 < 0.001
A x C 6 0.004 0.621 n.s. 6 0.011 0.850 n.s. 2 0.026 2.658 0.084
B x C 1 0.004 0.602 n.s. 1 0.001 0.047 n.s. 1 0.006 0.627 n.s.
A x B x C 6 0.001 1.738 0.118 6 0.002 0.129 n.s. 2 0.010 1.038 n.s.
Glumes type (D) 1 0.066 10.910 0.001 1 1.029 76.283 < 0.001 1 0.592 60.599 < 0.001
A x D 6 0.006 0.911 n.s. 6 0.005 0.407 n.s. 2 0.018 1.819 0.177
B x D 1 < 0.001 0.044 n.s. 1 0.008 0.620 n.s. 1 0.004 0.340 n.s.
A x B x D 6 0.005 0.905 n.s. 6 0.014 1.002 n.s. 2 0.012 1.261 n.s.
C x D 1 0.090 14.810 < 0.001 1 0.328 24.291 < 0.001 1 0.017 1.762 0.193
A x C x D 6 0.007 1.169 n.s. 6 0.002 0.111 n.s. 2 0.002 0.247 n.s.
B x C x D 1 < 0.001 0.004 n.s. 1 0.066 4.865 0.029 1 0.009 0.881 n.s.
A x B x C x D 6 0.003 0.445 n.s. 6 0.008 0.590 n.s. 2 0.012 1.234 n.s.
Error 126 0.006 126 0.013 36 0.010
Total 223 223 71
a
Mean square.
b
P values > 0.2 were indicated as not significant.
0
0
6
_
J
P
P
_
7
7
R
P
_
1
9
7


2
1
-
0
7
-
2
0
0
8


1
0
:
2
4


P
a
g
i
n
a

2
0
5
2
0
6
R
i
c
e

g
r
a
i
n

d
i
s
c
o
l
o
u
r
a
t
i
o
n
J
o
u
r
n
a
l

o
f

P
l
a
n
t

P
a
t
h
o
l
o
g
y

(
2
0
0
8
)
,

9
0
(
2
)
,

1
9
7
-
2
0
9
Table 7. Effect of chemicals, nitrogen fertilization and date of sowing on panicle brown spot, grain discolouration and milling yield of rice cv. Selenio at Rosasco (Pavia), Italy.
Panicle brown spot Grain discolouration (%) Milling yield (%)
Incidence (%) Severity (%)
Factor
1999 2000 2001 1999 2000 2001
1999 2000 2001 1999 2000 2001
Untreated control 21.25
b
n.s.
c
90.87 a
d
100 11.83 n.s.
c
62.52 a 21.54 n.s.
e
0.76 n.s. 1.90 n.s. 3.07 n.s. 60.32 n.s. 63.13 n.s. 55.21 n.s.
Tricyclazole 17.25 90.62 a 100 10.10 56.51 a 21.29 0.91 1.91 2.89 58.77 62.63 55.57
Azoxystrobin 16.62 78.12 b 100 8.73 34.80 b 20.00 0.70 1.94 3.32 60.68 64.73 57.39
Acibenzolar-S-methyl 16.12 90.00 a - 8.83 57.52 a - 0.75 1.98 - 59.79 61.50 -
Acibenzolar-S-methyl (2
treatments)
20.37 90.62 a - 10.97 62.37 a - 0.91 2.01 - 60.44 62.27 -
Tricyclazole + Acibenzolar-S-
methyl
15.00 - - 7.81 - - 0.91 - - 58.00 - -
Azoxystrobin + Acibenzolar-S-
methyl
18.37 - - 9.77 - - 0.83 - - 59.29 - -
Prohexadione-Ca 20.87 95.62 a - 12.44 67.05 a - 0.74 2.04 - 60.97 60.68 -
Prohexadione-Ca (2 treatments) - 96.25 a - - 66.28 a - - 1.65 - - 63.27 -
Chemicals
a
Prohexadione-Ca + Acibenzolar-
S-methyl
21.37 - - 11.92 - - 0.73 - - 61.26 - -
Low 19.00 n.s. 92.07 a 100 10.97 n.s. 63.65 a 22.47 a 0.78 n.s. 1.56 a 3.23 n.s. 59.97 n.s. 61.38 a 54.76 a N-fertilization
High 18.17 88.54 b 100 9.57 52.65 b 19.42 b 0.83 2.28 b 2.89 59.92 63.82 b 57.35 b
Early (Mid-April) 20.78 a 89.00 n.s. 100 11.86 a 55.43 a 20.86 n.s. 0.83 n.s. 3.01 a 4.03 a 58.75 a 57.87 a 53.13 a Date of sowing
Late (Mid-May) 16.39 b 91.61 100 8.67 b 60.87 b 21.03 0.78 0.82 b 1.88 b 61.13 b 67.33 b 58.97 b
a
See table 1 for details.
b
Mean value.
c
Duncans multiple range test P = 0.05 not significant.
d
Mean with the same letter are not significantly different.
e
ANOVA not significant.
0
0
6
_
J
P
P
_
7
7
R
P
_
1
9
7


2
1
-
0
7
-
2
0
0
8


1
0
:
2
4


P
a
g
i
n
a

2
0
6
J
o
u
r
n
a
l

o
f

P
l
a
n
t

P
a
t
h
o
l
o
g
y

(
2
0
0
8
)
,

9
0
(
2
)
,

1
9
7
-
2
0
9
P
i
z
z
a
t
t
i

a
n
d

C
o
r
t
e
s
i
2
0
7
Table 8. ANOVA to test the effects of chemicals, nitrogen fertilization and date of sowing on panicle brown spot, grain discolouration and milling yield of rice cv. Selenio at
Rosasco (Pavia), Italy.
1999 2000 2001 Factor Source
df MS
a
F P df MS
a
F P df MS
a
F P
Replication 3 0.089 15.091 < 0.001 3 0.277 8.298 0.001 n.d.
c
Chemicals (A) 8 0.018 3.067 0.016 6 0.185 5.533 0.002
Error 24 0.006 18 0.033
N fertilization (B) 1 0.007 0.653 n.s.
b
1 0.216 4.548 0.045
A x B 8 0.011 1.112 n.s. 6 0.024 0.508 n.s.
Error 27 0.010 21 0.047
Date of sowing (C) 1 0.139 28.718 < 0.001 1 0.087 1.556 n.s.
A x C 8 0.005 0.937 n.s. 6 0.09 0.160 n.s.
B x C 1 0.051 10.413 0.002 1 0.096 1.714 0.197
A x B x C 8 0.002 0.345 n.s. 6 0.034 0.606 n.s.
Error 54 0.005 42 0.056
Panicle brown spot
incidence
Total 143 111
Replication 3 0.052 12.985 < 0.001 3 0.251 15.813 < 0.001 2 0.005 3.855 0.117
Chemicals (A) 8 0.012 3.052 0.016 6 0.229 14.451 < 0.001 2 0.001 0.845 n.s.
Error 24 0.004 18 0.016 4 0.001
N fertilization (B) 1 0.022 3.556 0.070 1 0.428 19.374 < 0.001 1 0.013 23.299 0.003
A x B 8 0.005 0.736 n.s. 6 0.006 0.292 n.s. 2 0.002 2.962 0.127
Error 27 0.006 21 0.022 6 0.001
Date of sowing (C) 1 0.109 22.698 < 0.001 1 0.100 3.947 0.053 1 0.000 0.008 n.s.
A x C 8 0.003 0.557 n.s. 6 0.022 0.884 n.s. 2 0.004 2.094 0.166
B x C 1 0.015 3.143 0.082 1 0.056 2.196 0.146 1 0.017 10.129 0.008
A x B x C 8 0.002 0.323 n.s. 6 0.018 0.706 n.s. 2 0.001 0.385 n.s.
Error 54 0.005 42 0.025 12 0.002
Panicle brown spot severity
Total 143 111 35
Replication 3 0.002 3.942 0.020 3 0.017 25.580 < 0.001 2 0.001 1.340 n.s.
Chemicals (A) 8 < 0.001 0.570 n.s. 6 < 0.001 0.358 n.s. 2 < 0.001 0.267 n.s.
Error 24 0.001 18 0.001 4 0.001
N fertilization (B) 1 < 0.001 1.433 n.s. 1 0.021 21.862 < 0.001 1 0.001 3.754 0.101
Grain discolouration
A x B 8 < 0.001 1.320 n.s. 6 0.001 0.768 n.s. 2 < 0.001 0.584 n.s.
0
0
6
_
J
P
P
_
7
7
R
P
_
1
9
7


2
1
-
0
7
-
2
0
0
8


1
0
:
2
4


P
a
g
i
n
a

2
0
7
2
0
8
R
i
c
e

g
r
a
i
n

d
i
s
c
o
l
o
u
r
a
t
i
o
n
J
o
u
r
n
a
l

o
f

P
l
a
n
t

P
a
t
h
o
l
o
g
y

(
2
0
0
8
)
,

9
0
(
2
)
,

1
9
7
-
2
0
9
N fertilization (B) 1 < 0.001 1.433 n.s. 1 0.021 21.862 < 0.001 1 0.001 3.754 0.101
A x B 8 < 0.001 1.320 n.s. 6 0.001 0.768 n.s. 2 < 0.001 0.584 n.s.
Error 27 < 0.001 21 0.001 6 < 0.001
Date of sowing (C) 1 0.001 1.385 n.s. 1 0.175 117.435 < 0.001 1 0.044 76.480 < 0.001
A x C 8 < 0.001 0.403 n.s. 6 < 0.001 0.74 n.s. 2 0.001 1.810 0.206
B x C 1 0.001 1.849 0.179 1 < 0.001 0.015 n.s. 1 < 0.001 0.180 n.s.
A x B x C 8 < 0.001 0.900 n.s. 6 0.001 0.983 n.s. 2 0.001 0.912 n.s.
Error 54 < 0.001 42 0.001 12 0.001
Total 143 111 35
Replication 3 0.002 1.469 n.s. 3 0.017 12.670 < 0.001 2 0.001 1.096 n.s.
Chemicals (A) 8 0.002 1.162 n.s. 6 0.003 2.187 0.093 2 0.002 1.516 n.s.
Error 24 0.002 18 0.001 4 0.001
N fertilization (B) 1 < 0.001 0.010 n.s. 1 0.018 9.739 0.005 1 0.006 12.498 0.012
A x B 8 < 0.001 0.263 n.s. 6 0.002 1.318 n.s. 2 < 0.001 0.022 n.s.
Error 27 0.001 21 0.002 6 < 0.001
Date of sowing (C) 1 0.021 17.621 < 0.001 1 0.266 98.004 < 0.001 1 0.031 20.265 0.001
A x C 8 < 0.001 0.187 n.s. 6 0.002 0.584 n.s. 2 < 0.001 0.127 n.s.
B x C 1 0.004 3.173 0.080 1 0.001 0.239 n.s. 1 0.001 0.337 n.s.
A x B x C 8 0.002 1.282 n.s. 6 0.003 1.199 n.s. 2 < 0.001 0.161 n.s.
Error 54 0.001 42 0.003 12 0.002
Milling yield
Total 143 111 35
a
Mean square.
b
P values > 0.2 were indicated as not significant.
c
Not done.
0
0
6
_
J
P
P
_
7
7
R
P
_
1
9
7


2
1
-
0
7
-
2
0
0
8


1
0
:
2
4


P
a
g
i
n
a

2
0
8
REFERENCES
Baldacci E., Corbetta G., 1964. Ricerche sulla microflora delle
cariossidi di riso dopo conservazione in magazzino e in
condizioni sperimentali. Il Riso 13: 79-88.
Caufin A., Moletti M., 1988. Indagine riguardante i funghi
presenti sul risone alla raccolta nelle diverse zone risicole
italiane. 10 Convegno Internazionale sulla Risicoltura, Ver-
celli 1988: 271-278.
Cofelice G., Conca G., Infantino A., Riccioni L., Russo S.,
Porta-Puglia A., 2002. Osservazioni sulla microflora e le al-
terazioni cromatiche del risone e del riso lavorato. Informa-
tore Fitopatologico 52(2): 58-63.
Cortesi P., Bartoli F., Pizzatti C., Bertocchi D., Schaad N.W.,
2008. Panicle sterility and grain discolouration: new and
emerging bacterial diseases of rice in Italy. In: Fatmi M.B.,
Collmer A., Iacobellis N.S., Mansfield J.W., Murillo J.,
Schaad N.W. and Ullrich M. (eds.). Pseudomonas syringae
Pathovars and Related Pathogens-Identification, Epidemi-
ology and Genomics, pp. 391-411. Springer, Dordrecht,
The Netherlands.
Cortesi P., Bartoli F., Pizzatti C., Song W.Y., Schaad N.W.,
2005. First report of Acidovorax avenae subsp. avenae on
rice in Italy. Journal of Plant Pathology 87: 76.
Cortesi P., Giuditta L., 2003. Epidemiologia dellelmintospo-
riosi e del brusone e difesa del riso. Informatore Fitopato-
logico 53(2): 41-51.
Cottyn B., Cerez M.T., Van Outryve M.F., Barroga J., Swings J.,
Mew T.W., 1996. Bacterial diseases of rice. I. Pathogenic
bacteria associated with sheath rot complex and grain discol-
oration of rice in the Philippines. Plant Disease 80: 429-437.
Duveiller E., Miyajima K., Snacken F., Autrique A., Maraite
H., 1988. Characterization of Pseudomonas fuscovaginae
and differentiation from other fluorescent pseudomonads
occurring on rice in Burundi. Journal of Phytopathology
122: 97-107.
Duveiller E., Notteghem J.L., Rott P., Snacken F., Maraite H.,
1990. Bacterial sheath brown rot of rice caused by
Pseudomonas fuscovaginae in Malagasy. Tropical Pest Man-
agement 36: 151-153.
Fazli S.F., Schroeder H.W., 1966. Effect of kernel infection of
rice by Helmintosporium oryzae on yield and quality. Phy-
topathology 56: 1003-1005.
Friedrich L., Lawton K.A., Ruess W., Masner P., Specker N.,
Gut-Rella M., Meier P., Dincher S., Staub T., Uknes S.,
Kessmann H., Ryals J., 1996. A benzothiadiazole derivative
induces systemic acquired resistance in tobacco. The Plant
Journal 10: 61-70.
Giudici M.L., Villa B., 2006. Trigonotylus caelestialium
kirkaldy (Heteroptera, Miridae, Mirinae, Stenodemini) su
riso in Italia. Informatore Fitopatologico 56(6): 18-23.
Haegi A., Infantino A., Porta-Puglia A., 2002. Ruolo della
tossina di Bipolaris oryzae nelle alterazioni cromatiche del
riso. Informatore Fitopatologico 52(2): 52-55.
Infantino A., Pucci P., Di Giambattista G., Porta-Puglia A.,
1997. Seed health condition of rice seeds in Italy. In. 10
th
Congress of the Mediterranean Phytopatological Union,
Montpellier - Le Corum, 1997: 713-716.
Jahn C.G., Domingo I., Liberty M., Almazan P., Pacia J.,
2004. Effect of rice bug Leptocorisa oratorius (Hemiptera:
Alydidae) on rice yield, grain quality and seed viability.
Journal of Economic Entomology 97: 1923-1927.
Lancashire P.D., Bleiholder H., Van der Boom T., Langelud-
deke P., Stauss R., Weber E., Witzenberger A., 1991. A
uniform decimal code for growth stages of crops and
weeds. Annals of Applied Biology 119: 561-601.
Lawton K.A., Friedrich L., Hunt M., Weymann K., Kessmann
H., Staub T., Ryals J., 1996. Benzothiadiazole induces dis-
ease resistance in Arabidiopsis by activation of the systemic
acquired resistance signal transduction pathway. The Plant
Journal 10: 71-78.
Marchetti M.A., Petersen H.D., 1984. The role of Bipolaris
oryzae in floral abortion and kernel discoloration in rice.
Plant Disease 68: 288-291.
Moletti M., Giudici M.L., Villa B., 1996. Lakiochi-elmintospo-
riosi del riso in Italia: lotta agronomica e chimica. Informa-
tore Fitopatologico 46(6): 41-46.
Nissen O., 1983. MSTAT-C: a microcomputer program for
the design, management, and analysis of agronomic re-
search experiments. Michigan State University, East Lans-
ing, MI, USA.
Ou S.H., 1985. Rice Diseases. Commonwealth Mycological
Institute, Kew, UK.
Picco A.M., Lorenzi E., Rodolfi M., Biloni M., 2002. Mi-
cromiceti associati a quattro differenti variet di riso colti-
vate in Italia. Atti Giornate Fitopatologiche, Baselga di Pin
(Trento) 2002: 487-488.
Pizzatti C., 2005. Maculatura delle cariossidi del riso: eziolo-
gia della malattia, caratterizzazione della microflora batter-
ica e difesa. Tesi di Dottorato, Universit degli Studi di Mi-
lano, Milano.
Pizzatti C., Pedrali D., Cortesi P., 2004. Lack of correlation
between incidence of rice grain discolouration and panicle
brown spot. Journal of Plant Pathology 86: 329-330.
Rott P., Notteghem J.L., Frossard P., 1989. Identification and
characterization of Pseudomonas fuscovaginae, the causal
agent of bacterial sheath brown rot of rice, from Madagas-
car and other countries. Plant Disease 73: 133-137.
Ruess W., Mueller K., Knauf-Beiter G., Kunz W., Staub T.,
1996. Plant activator CGA 2450704: an innovative ap-
proach for disease control in cereals and tobacco. Proceed-
ings of the Brighton Crop Protection Conference, Pests and
Diseases, Brighton, 1996: 53-60.
Webster R.K., Gunnel P.S., 1992. Compendium of Rice Dis-
eases. APS Press, St. Paul, MN, USA.
Zeigler R.S., Alvarez E., 1987. Bacterial sheath brown rot of
rice caused by Pseudomonas fuscovaginae in Latin America.
Plant Disease 71: 592-597.
Zeigler R.S., Aricapa G., Hoyos E., 1987. Distribution of fluo-
rescent Pseudomonas spp. causing grain and sheath discol-
oration of Rice in Latin America. Plant Disease 71: 896-
900.
Zeigler R.S., Alvarez E., 1990. Characteristics of Pseudomonas
spp. causing grain discoloration and sheath rot of rice, and
associated Pseudomonad epiphytes. Plant Disease 74: 917-
922.
Journal of Plant Pathology (2008), 90 (2), 197-209 Pizzatti and Cortesi 209
Received September 3, 2007
Accepted December 28, 2007
006_JPP_77RP_197 21-07-2008 10:24 Pagina 209
006_JPP_77RP_197 21-07-2008 10:24 Pagina 210