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African Crop Science Journal
African Crop Science Society
ISSN: 1021-9730 EISSN: 2072-6589
Vol. 6, Num. 4, 1998, pp. 345-350
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African Crop Science Journal, Vol
African Crop Science Journal, Vol. 6. No. 4, pp. 345-350,
1998
Printed in Uganda. All rights reserved
Ó 1998, African Crop Science
Society
Screening techniques for Fusarium
wilt of pigeonpea
M.A. OKIROR
Department of Botany, Egerton University, P. O. Box 536, Njoro,
Kenya
(Received 4 August, 1997; accepted 21 December, 1998)
Code Number:CS98036
ABSTRACT
A repeatable inoculation procedure for Fusarium wilt
of pigeonpea caused by Fusarium udum Butler was developed.
Five techniques, namely, sowing seeds or transplanting seedlings
into infested soil, dipping roots or soaking seed in a spore
suspension, and stem injection were tested under glasshouse
conditions on four cultivars of pigeonpea with different levels of
resistance. Sowing seed in infested soils gave the highest
mortality and allowed for easy differentiation of resistant and
susceptible plants. The other techniques either gave severe
wilting, inconsistent results or low wilting, and were considered
unreliable. This study recommends that sowing seed in infested soil
in a glasshouse be adopted as a standard procedure for scoring this
disease.
Key Words: Cajanus cajan, Fusarium udum,
inoculation technique, root dipping, spore concentration, stem
injection
RÉSUMÉ
Une procédure d'inoculation périodique du
Fusarium wilt de Pigeonpea causé par le Fusarium
udum Butler a été développée. Cinq
techniques, notamment la semence de grains ou la transplantation
des semis dans un sol infecté, le trempage des racines ou
des grains dans une suspension de spores, et l'injection de la tige
étaient testées dans des conditions de serre sur
quatre variétés cultivées de pigeonpea aux
différents niveaux de resistance. La semence de grains dans
un sol infecté a donné une mortalité
très élevée et a permis une
différenciation aisée sur des plants
résistants et susceptibles. Les autres techniques ont
donné soit une sévère fanaison, soit des
résultats inconsistants ou un léger
dessèchement, et ont été
considérées comme moins confiantes. Cette
étude recommande que la semence des grains dans un sol
infecté placé dans des conditions de serre soit
adoptée comme une procédure modèle pour
combattre la maladie.
Mots Clés: Cajanus cajan, Fusarium udum,
techniques d'inoculation, trempage de racine, concentration de
spore, trempage de tige xxxx
INTRODUCTION
The success of an exercise to identify pigeonpea (Cajanus
cajan (L.) Millsp.) germplasm resistant to Fusarium wilt
caused by Fusarium udum Butler depends heavily on the
procedure used. Pigeonpea has traditionally been screened for wilt
resistance in wilt-infested fields (Butler, 1908; Deshpande et
al., 1963). The International Crops Research Institute for the
Semi-Arid Tropics (ICRISAT) also uses field screening but has
reported cases of inconsistent results (Nene et al., 1981).
They are also testing other techniques, especially for use in the
glasshouse.
Various techniques have been used for glasshouse screening for
resistance to soil-borne pathogens, including root dipping in
inoculum (Phipps and Stipes, 1973), growing seed or seedlings in
infested soils (Russell, 1978), soaking seed in inoculum (Sakar
et al., 1982) and injecting inoculum into plants (Jindal
et al., 1982). The effectiveness of these techniques depends
on various factors, including spore concentration, plant age at
inoculation, and environmental conditions such as temperature and
humidity (Ribeiro and Hagedorn, 1979). Plant injury, especially to
the roots, enhances infection (Henderson and Winstead, 1961). The
objective of this study was to find a suitable glasshouse technique
for screening pigeonpea germplasm for resistance to Fusarium
wilt.
MATERIALS AND METHODS
Cultivars. Four pigeonpea cultivars - NPP 679, ICP 2376, ICP
2376, ICP 270 and Munaa - were used in this study. Two of these,
NPP 679 and Munaa, are local cultivars. The former is resistant to
Fusarium wilt while the latter is susceptible. The other
two, ICP 2376 and ICP 270 are from ICRISAT and are susceptible and
resistant to wilt, respectively. All these cultivars had been
selfed for four generations and were considered genetically
homozygous.
Inoculum. Inoculum was prepared from wilted pigeonpea
plants collected from Thika, a non-traditional pigeonpea growing
area in the Kiambu district of central Kenya. Plant roots and stems
were chopped and incorporated into large wooden boxes (94 x 50cm x
43cm deep) filled with forest soil in the glasshouse. One kg of
plant material per 5 kg soil was put in the boxes. The boxes were
watered lightly everyday, and more plant material was added at the
rate of 1kg box-1 monthly until the boxes were confirmed to be
infested with Fusarium. They were then designated sickboxes.
Single spore cultures of F. udum were produced on potato
dextrose agar (PDA), and used to prepare inoculum suspensions.
Inoculation techniques. Five techniques were compared,
namely, (a) sowing seed in infested soil, (b) transplanting
seedlings into infested soil, (c) soaking seed in inoculum, (d)
dipping roots of seedlings in inoculum, and (e) stem injection of
inoculum.
(a) Sowing seeds. Thirty seeds per cultivar were sown in
a sickbox. Furrows were marked 7 cm apart across the box. Each
cultivar was sown in three furrows (rows) at a rate of ten seeds
per furrow. Non-infested soil in a similar box was used as the
control.
(b) Transplanting into sick boxes. Twenty three-weeks old
seedlings per cultivar grown in Fusarium-free soil were
used. Roots were trimmed on ten seedlings but were left intact on
the other ten. Seedlings were transplanted into furrows in the
sickbox. Three furrows of seedlings were planted, each with intact
and trimmed roots per cultivar. Seedlings treated similarly were
planted in pathogen-free soil.
(c) Seed soaking. Four spore concentrations were used:
4.2 x 105, 9.4 x 105, 1.3 x 106 and 1.6 x 106 spores ml-1. Thirty
seeds per cultivar were placed in a beaker containing about 200 ml
of each of the inocula. At 1, 5 and 7 h after immersion, ten seeds
were removed from each beaker and sown in a furrow in disease-free
soil. Seeds soaked in water served as the control.
(d) Root dipping. Twenty three-weeks old seedlings of
each cultivar were tested at each spore concentration used for seed
soaking. Of these ten had their roots trimmed. Seedling roots were
immersed in one of the four spore concentrations for 1.5 hr and
transplanted into non infested soil in the boxes. Seedlings with
roots dipped in water served as a control.
(e) Stem injection. Two spore concentrations were used:
1.3 x 106 and 1.6 x 106 spores ml-1, and control seedlings received
water. 0.25 ml of inoculum in a hypodermic syringe was injected
into a four-weeks old seedling, 4 cm above ground. Ten seedlings
per cultivar were injected with each inoculum.
Methods (a) and (b) were repeated three times each while methods
(c), (d) and (e) were repeated four times. All experiments were
conducted in a glasshouse at the University of Nairobi, Kabete
Campus. Day temperatures in the glasshouse were 38-42$C (maximum)
and 25-29$C (minimum). Initial plant stands were recorded 14 days
after either germination (if seed were sown), or when transplanted
seedlings had survived (in case of transplants).
Data collection and analysis. The onset of wilting and
the number of plants wilted was recorded weekly; final counts were
taken three months after inoculations.
RESULTS
Characteristic wilt symptoms were observed where F.
udum inoculum was used. No wilting appeared until at least four
weeks after inoculation, and at least nine weeks after stem
injection.
Direct sowing. The first wilted plant appeared four weeks
after inoculation in Munaa, and was followed rapidly by more deaths
in both Munaa and ICP 2376. Wilt was slow to occur and spread in
ICP 270. NPP 679, originally considered resistant, broke down and
was heavily wilted. The overall mean wilt caused by this technique
was 56% and was the highest in all the methods studied. Susceptible
and resistant cultivars were easily distinguished (Table 1).
Transplanting seedlings into sick boxes. Wilted plants
first appeared in the fifth week in both Munaa and ICP 2376. By the
end of week seven, the extent of wilting was much higher and all
cultivars were affected, but the susceptible cultivars were more
severely affected and distinguishable from the resistant cultivars.
Again, NPP 679 was heavily affected. The average mortality was 46%
for seedlings with intact roots and 51% for those with pruned
roots; this difference was not statistically significant (Table 1).
This technique was ranked third after direct sowing and root
dipping.
Stem injection. No wilt was recorded until the ninth week
when one plant of Munaa wilted. This plant had received the higher
of the two inoculum concentrations. Eventually, a few wilted plants
appeared in all of the cultivars. Affected plants were generally
only partially wilted. This technique caused the least wilting
(Table 1).
TABLE 1. Effect of inoculation method on
Fusarium wilt incidence in four pigeonpea cultivars (% of
plants wilted)
Cultivar |
Direct sowinga |
Inoculation techniques |
Seedlings transplanting |
Stem injection |
Root treatment |
Spore concentrationb |
Intact |
Pruned |
3 |
4 |
Munaa |
88.9 |
57.0 |
58.3 |
25.0 |
20.0 |
NPP679 |
38.8 |
48.3 |
52.3 |
4.9 |
6.8 |
NPP718 |
82.0 |
63.3 |
68.0 |
15.3 |
177.7 |
NPP725 |
12.5 |
16.2 |
24.4 |
2.4 |
3.3 |
Method mean |
55.7 |
46.2 |
50.8 |
11.9 |
12.0 |
CV (%) |
30.4 |
12.0 |
40.4 |
? |
? |
a =% of plants wilted
bSpore concentrations: 1 = 4.2 x 105, 2= 9.4 x 106, 3 =
1.3 x 106, 4 = 1.6 x 106 spores ml-1
Root dipping. Like direct sowing, root dipping also led
to onset of wilting four weeks after inoculation. The plants that
wilted had their roots trimmed and dipped in the most concentrated
inoculum. Wilting increased with time and in all cultivars and root
treatments. By the end of the study, more wilted plants were
recorded, especially at the highest inoculum level. At the three
highest levels of inoculum separation of resistant and susceptible
cultivars was difficult (Table 2). However, at the lowest inoculum
concentration, resistant and susceptible cultivars reacted
differently and distinctly although all mortalities were lower.
Root injury did not give any major advantage over intact roots.
TABLE 2. Effect of root dipping seedlings
on wilt incidence in four cultivars of pigeonpea (% of plants
wilted)
Cultivar |
Root treatment |
Spore concentrations |
1 |
2 |
3 |
4 |
Munaa |
Intact |
46.7 |
50.9 |
64 |
90.0 |
Pruned |
43.5 |
54.8 |
74 |
85.5 |
NPP 679 |
Intact |
22.2 |
43.0 |
47 |
52.8 |
Pruned |
26.5 |
45.7 |
50 |
55.0 |
ICP 2376 |
Intact |
50.0 |
55.5 |
60 |
83.0 |
Pruned |
50.0 |
59.5 |
65 |
79.4 |
ICP 270 |
Intact |
12.5 |
43.8 |
45 |
60.0 |
Pruned |
15.0 |
40.0 |
44 |
65.5 |
Means |
Intact |
32.9 |
48.3 |
54 |
71.0 |
Pruned |
33.8 |
50.0 |
58 |
71.4 |
CV (%) |
4.4 |
a Spore concentrations: 1 = 4.2 x
105, 2 = 9.4 x 105, 3 = 1.3 x 106, 4 = 1.6 x 106 spores
ml-1
Seed soaking. For all cultivars, wilting increased with
increasing spore concentration and/or length of soaking. The mean %
wilted plants for 1, 5, and 7 hr of soaking were 19, 19 and 26,
respectively, and 13, 17, 23 and 28 for the four inoculum
concentrations. This technique generally induced less wilting than
the previous two methods (Table 3).
TABLE 3. Effect of soaking seed in
different inoculum concentrations on wilt incidence in four
cultivars of pigeonpea (% of plants wilted)
Cultivar |
Time (hr) |
Spore concentrations |
1 |
2 |
3 |
4 |
Munaa |
1 |
5.6 |
4.8 |
14 |
14.3 |
5 |
13 |
20 |
16 |
21.4 |
7 |
25 |
18.5 |
25 |
42.9 |
NPP 679 |
1 |
8.8 |
8.6 |
5.4 |
9.1 |
5 |
9.5 |
23.5 |
2.7 |
33.3 |
7 |
16 |
13.2 |
13 |
27.8 |
NPP 718 |
1 |
10 |
12.5 |
25 |
30 |
5 |
22 |
23.2 |
33 |
33.3 |
7 |
11 |
40 |
43 |
50 |
N PP 725 |
1 |
11 |
3.5 |
33 |
40 |
5 |
13 |
14 |
20 |
10 |
7 |
13 |
17.4 |
40 |
20 |
Means |
1 |
8.9 |
7.4 |
19 |
23.4 |
5 |
14 |
20.2 |
18 |
24.5 |
7 |
16 |
22.3 |
30 |
35.2 |
CV (%) |
4.4 |
a Spore concentrations: 1 = 4.2 x
105, 2 = 9.4 x 105, 3 = 1.3 x 106, 4 = 1.6 x 106 spores
ml-1
Discussion
Several methods of inoculating pigeonpea with
Fusarium were compared. Sowing seed directly into infested
soil was the most effective. Wilting occurred early in the plant's
growth, and the level of wilting induced was sufficient to
differentiate between resistant and susceptible cultivars.
Moreover, the technique is simple, reliable, easy to apply, and
cost-effective and corresponds to sowing in infested soil in the
field (Hubbeling, 1980). This technique has been reported to give
the best comparison with field results in screening for resistance
to Fusarium oxysporum f. vasinfectum in cotton
(Hillocks, 1984). However, it is difficult to quantify the inoculum
in the soil.
The root dip method resulted in a high percentage of wilted
plants in all cultivars, especially at the three highest inoculum
levels. However, it was considered more reliable at the low
inoculum levels since the difference between susceptible and
resistant cultivars was more apparent. Similar results with this
technique have been reported by Phipps and Stipes (1973) for
Mimosa. Root pruning was expected to predispose plants to
early fungal invasion and lead to heavier wilting; however, the
results showed no such a trend. This method was labour-and
time-intensive and it may not be useful for screening large numbers
of plants.
Wilting started late and progressed slowly in plants grown from
seed soaked in various inoculum suspensions. The susceptible
cultivars had 5 to 50% wilted plants while the resistant cultivars
had 4 to 40%. This method, therefore, does not clearly separate
resistant and susceptible pigeonpea plants.
Raising seedlings and then transplanting them into infested soil
offered no signicant advantage over direct sowing of seed in such
soil. It neither led to earlier occurrence of wilt nor induced
higher levels of wilting even when seedlings had their roots
pruned. Given that this technique requires equipment to grow
seedlings, culture the pathogen, prepare and standardise the
inoculum, it is not as useful as the direct sowing techique
discribed above.
The results of stem injection suggest that it is not an
effective method of screening as previously observed by Sharma
et al. (1977). Besides inducing only very low wilting, it is
slow and requires significant labour to inject the plants. Of the
methods studied, stem inoculation was the least effective.
In conclusion, sowing seed into infested soil is the most
effective approach for screening pigeonpea for resistance against
F. udum. This technique is easy to carry out, gives
dependable results and is similar to field screening except for
controlled conditions. Root dipping and transplanting seedlings
into infested soil, are also effective but have logistical
shortcomings that make direct sowing of seed a most appropriate
technique. Both seed soaking and stem injection were unreliable.
For the immediate needs of screening pigeonpea germplasm for
resistance to Fusarium wilt, glasshouse screening shall be
adopted, based on the sickbox test.
REFERENCES
- Butler, E.J. 1908. Selection of pigeonpea for wilt disease.
Agricultural Journal of India 3: 182-183.
- Deshpande, R.B., Jeshwani, L.M. and Joshi, A.B. 1963. Breeding
of wilt resistant varieties of pigeonpea. Indian Journal of
Genetics and Plant Breeding 23:58-63.
- Henderson, W.R. and Winstead, N.N. 1961. Reaction of tomato
varieties and breeding lines to Fusarium oxysporum f.
lycopersici Race 1. Plant Disease Reporter
45:272-273.
- Hillocks, R.J. 1984. Production of cotton varieties with
resistance to Fusarium wilt with special reference to
Tanzania. Tropical Pest Management 30:234-246.
- Hubbeling, N. 1980. Laboratory/glasshouse screening for
identifying resistance to soil-borne diseases in beans. Pages
123-128. In: Proceeding of Consultants' Group Discussion on the
Resistance to Soil-borne Diseases of Legumes, 8-11 January
1979, Hyderabad, India. ICRISAT, India.
- Jindal, J.K., Patel, P.N. and Khan, A.M. 1982. Variability in
Xanthomondads of grain legumes. II. Pathogenic variability in
Xanthomonas phaseoli mungbean strains X. vignicola
and X. phaseoli var. sojense. Phytopathology Z.
100:1-9.
- Nene, Y.L., Kannaiyan, J., Reddy, M.V. and Remanandan, P. 1981.
Sources of resistance to selected pigeonpea diseases. Pulse
Pathology Progress Report No. 16. Patancheru, A.P. 502 324,
ICRISAT, India. 34pp.
- Phipps, P.M. and Stipes, R.J. 1973. Artificial reproduction of
Fusarium wilt of the Mimosa tree under greenhouse and
field conditions. Phytopathology 63:804 (Abstract).
- Ribeiro, R.L.D. and Hagedorn, D.J. 1979. Screening for
resistance to and pathogenic specialization of Fusarium
oxysporum f. sp. phaseoli, the causal agent of bean
yellows. Phytopathology 69:272-276.
- Russell, G.E. 1978. Variability in fungal pathogens. In:
Plant Breeding for Pest and Disease Resistance. Russell,
G.E. (Ed.), pp. 52-59. Butterworths and Co., London.
- Sakar, D., Muehlbauer, F.J. and Kraft, J.M. 1982. Techniques of
screening peas for resistance to Phoma medicaginis var.
pinodella. Crop Science 22:988-992.
- Sharma, N.D., Joshi, L.K. and Vyas, S.C. 1977. A new stem
inoculation technique for testing Fusarium wilt of
pigeonpea. Indian Phytopathology 30:406-407.
Copyright 1998, African Crop Science Society
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