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.Zbl. Bakt. Abt. II, Bd. 132, S. 75-80 (1977)
[Department of Botany, Ranchi University Ranchi, India]
Application and Standardization of Various Procedures for Inoculation of :Maize by Erwinia carotovora f. sp. zeae
Mahendra Prasad and S. K. Sinha
Summary
Hypodermic syringe and toothpick methods were best for evaluating pathogenicity of the maize plant against Erwinia carotovora f. sp. zeae, both in glass house and in the field. The portion of the stalk above the ground was best suited for reproducing the disease symptoms. Leaf and leaf whorl did not show any rotting here. Seed inoculation showed least mortality percentage. Root inoculation was suitable only for young seedlings. Cotyledons, as an organ for inoculation for getting best reproducible infection, were not at all suitable.
Zusammenfassung
Sowohl in Gewachshaus- als auch in Feldversuchen war die Anwendung der "hypodermatischen Spritze" und die "Zahnstochermethode" am besten fiir die Beurteilung der Anfalligkeit von Mais gegeniiber Erwinia carotovora f. sp. zeae geeignet. Am iiber dem Boden befindlichen Teil des Stengels JieJ3en sich die Krankheitssymptome am besten reproduzieren. Blatt und Blattquirl zeigten hier kein Krankheitsbild. Beimpftes Saatgut zeigte die geringste Absterberate. Wurzelinokulierung war nur fUr junge Setzlinge geeignet. Die Kotyledonen, Organe, die Infektionen am besten reproduzieren, waren iiberhaupt nicht geeignet.
Inoculation techniques vary, and it is essential to standardize this important procedure before investigations on all dimensions of disease incidence and control, specially epidemiological aspects and assessment of resistant germ plasm, can be done. Reproduction of symptoms, to a large extent, depends upon the efficiency of a particular inoculation method. Bacterial stalk rot of maize, caused by Erwinia carotovora f. sp. zeae Sabet (also known as Erwinia chrysanthemi, DYE 1969; recently being re-designated as Pectobacterium chrysanthemi pathovar zeae, KELMAN 1974), is a destructive rainy season maize crop disease in India (HINGORANI, GRANT, and SINGH 1959; LAL, THIND, PAYAK, and RENFRO 1970). Introduction of new inoculation techniques and their comparative evaluation for maize diseases has been made from time to time by YOUNG (1943), WERNHAM (1949), KOEHLER (1950, 1960), OAPPELLINI (1959), RANGARAJAN and OHAKRAVARTI (1970), HARTMAN and KELMAN (1973), and DIWAKAR and PAYAK (1974). A comparative study of varied inoculation techniques in regard to bacterial stalk rot of maize is presented here.
Materials and Methods
For glass house (G.H.) studies, maize seeds of hybrid Ganga-5 were raised in sterilized soil in plastic pots of different size at the rate of 5 plants per pot. Four isolates, two from Pantnagar, U.P.; one each from Udaipur, Rajasthan and Dhaulakuan, H.P., were used for inoculation. These isolates were pretested for pathogenicity on 21-day old maize plants and were found to be highly virulent.
76 M. PRASAD and S. K. SINHA
The plants were inoculated at the age of 30, 45, and 60 days, unless stated otherwise in the table. All inoculated plants were kept in a humid chamber for a week at 35°C and 70 per cent humidity with 12 hours' photoperiod.
Field inoculations (F.T.) were done under natural conditions. In case of hypodermic syringe method, 2 X 108 cells/ml of bacterial suspension was used in both glass house as well as field trials for all age groups of plants. 200 plants were inoculated in each treatment, and for root and cotyledons 100 seeds were used in each test.
A) Stalk inoculation
1. Hypodermic syringe: 1 ml of actively growing bacterial culture was injected into the basal portion of the stalk by sterilized hypodermic syringe of 20 ml capacity with 15 gauge needle.
2. Toothpick: Round wooden toothpicks, about 6 cm long, were used. The above toothpicks were first sterilized. They were washed several times in tap water before boiling to remove all toxic substances that inhibit growth of bacteria. Boiling was done in water several times, each time for one hour, before they were washed thoroughly with fresh tap water. They were then packed into glass jars and enough distilled water was added to moisten the toothpicks completely before sterilization. Then all the jars containing tootphicks were sterilized immediately at 20 lb. pressure for half an hour. 'l'hese sterilized toothpicks were properly mixed with a thick paste of 24 hours actively grown bacterial culture.
Tht inoculum was then ready for immediate use for glass house and field trials. For inoculating maize plants by the toothpicks, the desired hole was made into the first or second internode of the plant with an iron nail and then the toothpick was inserted into the hole at an angle. The iron nail was prepared by fixing a 2" nail to the end of a wooden handle, about 4" in length and 1" in diameter.
3. Multi-needle: The stalk of the plant was inoculated with the aid of a multi-needle, charged with bacterial culture_ The multi-needle outfit was prepared by fixing 1" pins with their heads embedded at one end of the wooden handle, about 4" in length and lit in diameter. 20 pins were fixed uniformly to provide a multi-needle pricking unit.
B) Underground stalk inoculations
The soil, covering the crown of the stem, was removed and the basal internodes were inoculated with the aid of a needle charged with bacterial culture. The point of inoculation was smeared with vaspar (1: 1) and covered with soil again.
C) Leaf inoculation
48 hours' actively growing bacterial culture suspension was sprayed on the leaf by a hand sprayer for inoculating the leaf.
D) Leaf whorl inoculation
1 ml of 48 hours' actively growing bacterial culture of 2 X 108 cells/ml strength was poured into leaf whorl of the plants to achieve inoculation.
E) Cob inoculation
The cob inoculation was done in the field only at the time of silking. The silk of the cob was cut with sterile scissors and 1 ml of the bacterial suspension, containing 2 X 108 cells/ml, was introduced into the cob through cut portions of the silk by the help of a sterilized pipette. The cobs were, after obtaining inoculation, covered with polythene bags.
F) Ear inoculation
The ear inoculation was also done in the field. 70 to 75-day old plants, where grains were formed, were inoculated by a similar method applied for the cob.
G) Root inoculation
100 seeds of maize hybrid Ganga-5 variety were germinated in agar plates in the laboratory. After the seeds had germinated and root growth had taken place, the root tips were cut with sterilized scissors and the plants were kept in a flask, containing 2 X 108 cells/ml bacterial suspension for 6 hours. After this the plants were transferred into pots containing autoclaved soil.
Application and Standardization of Various Procedures 77
H) Seed Inoculations
lOO seeds of Ganga.hybrid 5 variety were soaked in bacterial suspension for 12 hours. These soaked seeds were sown into pots containing sterilized soil and watered with sterile distilled water. In about 5 to 6 days, or when the plants began to emerge, the pots were placed in a moist chamber to prevent excessive drying.
I) Cotyledon inoculation
100 seeds were germinated on agar plate in a well illuminated laboratory. The cotyledons of germinated seeds were inoculated with 48 hours' actively growing bacterial culture with the help of a sterilized fine needle by pricking. Cotyledons, without being inoculated but similarly pricked, were kept as control.
Besides this, other additional tests were conducted with cotyledons of the same seed, using agar cultures on nonpathogenic Escherichia coli and Bacillus subtilis and pathogenic Erwinia carotovora f. sp. zeae. This was done for determining the susceptibility of the cotyledons to inoculation by known maize pathogenic and non.pathogenic bacteria. The cotyledons were inoculated by the same methods as mentioned above. The control was maintained under similar conditions.
Results and Discussion
A detailed list of all the methods employed along with the age group of plants, their incubation period, and percentage of mortality in the glass house as well as in the field, are included in Table 1. As is evident from this table among the different methods used, hypodermic syringe and toothpick methods were found to be more suitable and better than the others.
In case of leaf and leaf whorl inoculation, no infection at all could be observed. HOPPE and KELMAN (1969) could get infection in the field in dent corn single cross WF 9 X 22 by simply pouring bacterial suspensions into the whorl. HARTMAN and KELMAN (1973) have further evolved and introduced an improved method of whorl inoculation with 0.7 % Tween 40 surfactant. This method was tried here. May be due to the absence of Tween 40 in our case, leaf whorl inoculation did not produce any infection. According to HARTMAN and KELMAN (1973), use of Tween 40 along with the suspension of the pathogen Erwinia chrysanthemi (ECZ), the causal organism of the sweet corn, not only enhanced and stabilized infection, but with progressive increase in concentration of this surfactant a parallel stimulation in rotting of the plants could be achieved.
Cotyledon, as an organ for inoculation, was not found satisfactory, since it gave false results in regard to the size, shape and colour of the lesions developed both with the pathogenic and non· pathogenic bacteria.
RANGARAJAN and CHAKRAVARTI (1970) have also reported different methods of inoculation. Some of their methods, like multiple needle, root and seed inoculation employed here, were not found as effective as in their reports. The methods were, therefore, modified and then applied to the present studies.
In case of multiple needle, the inoculum was introduced inside the stalk with the aid of a multiple needle charged with bacterial culture, contrary to the practice of RANGARAJAN and CHAKRAVARTI (1970) who had simply placed the bacterial culture on a previously wounded surface made by multiple needle. There is a chance in such a case that the inoculum does not enter inside the host tissue thus giving wrong impression.
Root inoculation was found effective here, but could be applicable only for young seedlings. Germinated seedlings, previously raised on agar plates, were used here for inoculations. The younger plants can be transplanted easily as compared to older plants. RANGARAJAN and CHAKRAVARTI (1970) have used one month old plants for their inoculation studies. One month old uprooted plants, after injury, cannot be
Tab
le 1
. D
iffe
ren
t p
roce
du
res
emp
loy
ed f
or
ino
cula
tin
g m
aize
pla
nts
wit
h E
rwin
ia c
arot
ovor
a f.
sp
. ze
ae
-:J
00
Met
ho
d o
f in
ocu
lati
on
A
ge
of
Per
cen
tag
e o
f p
lan
ts i
nfe
cted
In
cu
bati
on
th
e
Dif
fere
nt
iso
late
s u
sed
l)
peri
od
in
day
s F=
pla
nt
'"d
84
(I-
I)
88 (
1·5)
8
9 (
1-6)
9
3 (
1-10
) ~ 00
in d
ay
s G
.H.
F.T
. G
.H.
F.T
. G
.H.
F.T
. G
.H.
F.T
. G
.H.
F.T
. >- lj '"
Ino
cu
lati
on
of
sta
lk (
ab
ov
e g
rou
nd
) ~ ~
a)
Hy
po
der
mic
sy
rin
ge
30
1
00
85
9
5
80
1
00
8
5
10
0
85
3
-5
5-
7 !Z
l 4
5
75
60
7
0
60
7
5
65
7
5
60
4
-6
6-
9 pi
6
0
35
4
0
52
4
5
56
4
5
53
4
5
5-
7 7
-1
0
rn
H Z
b)
To
oth
pic
k
30
95
85
9
0
80
9
5
80
9
0
80
5
-7
7-
9 ~ >-
45
7
0
60
7
0
60
7
2
64
7
5
58
6
-9
9-1
2
60
5
5
45
5
0
45
5
5
45
5
5
45
7
-1
0
12
-15
c)
Mu
lti-
nee
dle
s 3
0
65
4
5
60
5
0
65
5
0
60
4
5
6-
9 7
-lO
4
5
40
3
0
40
3
5
45
3
5
40
3
0
7-1
0
9-1
2
60
30
2
0
25
2
0
30
2
5
25
2
0
8-1
2
12
-15
Ino
cu
lati
on
of
sta
lk (
un
de
r g
rou
nd
)
Nee
dle
ch
arge
d w
ith
bacte
rial
30
6
0
45
6
5
50
6
5
30
6
0
45
5
-7
7-1
0
cult
ure
4
5
40
35
4
5
40
4
5
40
4
0
35
7
-1
0
9-1
2
60
25
2
0
25
2
0
25
2
0
25
2
0
7-1
0
12
-15
Ino
cula
tio
n o
f le
af
21
No
in
fect
ion
Ino
cula
tio
n o
f le
af w
ho
rl
21
No
in
fect
ion
Ino
cula
tio
n o
f co
b
50
-55
95
9
0
95
9
0
7-1
2
Ino
cula
tio
n o
f ea
r 7
0-7
5
95
95
1
00
9
5
lO-lo
Ino
cula
tio
n o
f ro
ots
7
-10
5
0
55
5
5
50
5
-7
Ino
cula
tio
n o
f se
eds
12 h
ou
rs
25
25
2
5
30
7
-lO
Ino
cula
tio
n o
f co
tyle
do
ns
5 -
7 F
alse
res
ult
3
-5
G.H
. =
G
lass
h
ou
se
ino
cula
tio
n.
F.T
. =
F
ield
in
ocu
lati
on
. -
=
No
in
ocu
lati
on
s m
ad
e.
84
(I-
I) =
U
daip
ur,
R
aja
sth
an
, 8
8 (
1-5)
=
Pan
tnag
ar,
U
ttar
Pra
des
h.
89 (
I-6
) =
P
an
tnag
ar,
Utt
ar
Pra
des
h.
93 (
I-lO
) =
D
hau
lku
an
, H
imach
al
Pra
desh
. 1)
Is
ola
tes
ob
tain
ed b
y c
ou
rtes
y o
f P
rofe
sso
r D
r. A
. K
EL
MA
N.
Un
iver
sity
of
vV
isco
nsi
n,
Mad
iso
n,
Wis
con
sin
U.S
.A.
Application and Standardization of Various Procedures 79
transplanted so easily. They had also kept injured roots for 24 hours in bacterial culture suspension. This gives an equal chance of rotting of the root even in nonpathogenic bacterial suspension. In order to avoid this risk, young germinated seedlings from agar plates were used here. Besides this the injured roots were dipped into bacterial suspension only for 6 hours before transplanting, which is less time-consuming.
Seed inoculation was done by soaking the seeds for 12 hours in bacterial suspension before hand, so that the natural seed rotting in the soil may not have interfered with our purposes. RANGARAJAN and CHAKRAVARTI (1970) soaked the wounded and nonwounded seeds for 24 hours in suspension. This is unnecessary as 12 hours' soaking is sufficient to cause infection. Again, it is not essential to wound the seeds siI).ce unwounded seeds also give similar results if kept for 24 hours' in bacterial suspension. The chances of rotting of wounded seed in soil as such are higher after sowing. With the avoidance of all such factors that may cause natural rotting of the seed, here it was found that the seed inocuJation yielded least mortality, thus suggesting the negative role of seed in disease transmission. This fact is also corroborated by serological studies whose results will be published elsewhere.
The toothpick method of inoculation, as tried here, was developed by YOUNG
(1943) and KOEHLER (1950, 1960) which is now being used extensively for inoculation of the fungal pathogens. DIWAKAR and PAYAK (1974) found the toothpick method to be superior to several other methods tried for evaluating the different inoculation techniques in regard to stalk rot of maize, caused by Pythium aphanidermatum Edson (Fitzp.). This method, when tried here for the inoculation of E. carotovora f. sp. zeae in maize stalk, was found to be as good as the hypodermic syringe method, both in glass house and field studies, provided that the sterilized toothpick is mixed thoroughly with a thick paste of 24 hours' grown bacterial culture, obtained after the inoculation of potato slices and not from the agar culture directly, and also that it is used immediately for inoculation.
Acknowledgements
l \We are extremely grateful to Professor Dr. A. KELMAN, University of Wisconsin, Madison, Wisconsin, U.S.A., for sending us the four bacterial isolates. Dr. KELMAN had obtained these cultures from Dr. M. M. PAYAK ofLA.R.L New Delhi, to whom we also extend our thanks. We are also obliged to Professor Dr. Y. L. NENE, G. B. Pant University of Agriculture and Technology, Pantnagar, U.P., for providing the necessary glass house facilities. We extend our thanks to Professor Dr. J. P. SINHA, Head of the Department of Botany, Ranchi University, for giving part facilities.
Literature
CAPPELLINI, R. A.: A comparison of techniques and sites of inoculation in field corn, artificially inoculated with Gibberella zeae (Schw.) Petch. Plant Dis. Rept. 43 (1959) 177 -179.
DIWAKAR, M. C., and PAYAK, M. M.: Evaluation of different inoculation techniques to induce Pythium stalk rot of maize. Sci. and Cult. 40 (1974), 431-433.
DYE, D. W.: A taxonomic study of the genus Erwinia. II. The "Carotovora" group. N.Z.J. of Sci. 12 (1969),81-97.
HARTMAN, J. R., and KELMAN, A.: Improved method for the inoculation of corn with Erwinia sp. Phytopathology 63 (1973),658-663.
HINGORANI, M. K., GRANT, U. J., and SINGH, N. J.: Erwinia carotovora f. sp. zeae, a destructive pathogen of maize in India. Indian Phytopath. 12 (1959), 151-157.
HOPPE, P. E., and KELMAN, A.: Bacterial top and stalk rot disease of corn in Wisconsin. Plant Dis. Rept. 53 (1969),68-70.
KELMAN, A.: Personal communication (1974).
80 M. PRASAD and S. K. SINHA, Application and Standardization of Various Procedures
KOEHLER, B.: Corn stalk and ear rot studies. Improved techniques in hybrid seed corn production. Tept. 5th Hybrid Corn Ind. Res. Conf. Amer. Seed Trade Assoc. Publ. 5 (1950),33-46.
- Corn Stalk rot in Illinois. Illinois Agri. Expt. Sta. Bull. 658 (1960). LAL, S., THIND, B. S., PAYAK, M. M., and RENFRO, B. L.: Bacterial stalk rot of maize in resistance
breeding and chemical control. Indian Phytopath. 23 (1970),156-157. RANGARAJAN, M., and CHAKRAVARTI, B. P.: Bacterial stalk rot of maize in Rajasthan, effect on
seed germination and varietal susceptibility. Indian Phytopath. 23 (1970),470-477. WERNHAM, C. C.: Techniques for inoculating corn with disease.producing organisms. Penn. Agr.
Expt. Sta. Prg. Rept. 5 (1949),1-17. YOUNG, H. C. (jr.): The toothpick method of inoculating corn for ear and stalk rot. Phytopathology
33 (1943), 16 (Abstr.).
Authors ' addresses:
Dr. MAHENDRA PRASAD, Department of Botany Ranchi, University, Ranchi-834008 (Bihar), India, and Dr. S. K. SINHA, Ranchi Agricultural College, Department of Plant Pathology, Rajendra Agricultural University, Camp.Kanke (Ranchi) (Bihar), India.
