The chickpea is one of the most important pulse crop grown through out India. Area and production are decreasing in Punjab due to uncertainty and risk involved in its cultivation due to occurrence of several diseases. Chickpea wilt caused by Fusarium oxysporum f.sp. ciceri is one of the limiting factors in its cultivation. Traditionally, the use of toxic chemicals was the common tool for reducing the losses due to plant diseases. However, in recent years concern has increased regarding hazardous effects of chemicals on the life of man and animals. The intensive use of chemicals has also lead to the increase in pathogen resistance to these chemicals. Such problems have instigated the search for the alternative approaches like biocontrol in the management of diseases.
Among the biocontrol agents, the nonpathogenic isolates of F. oxysporum and fluorescent Pseudomonas are common soil inhabitants and have been widely used as antagonists against fusarial wilt of several agricultural important crops (Alabouvette et al., 1998; Alabouvette and Steinberg, 1995; Duijff et al., 1998; Fuch et al., 1997, 1999; Paul et al., 1999).
In preliminary studies the selected isolate Fo52 of nonpathogenic F. oxysporum
and isolate Pf5 of fluorescent Pseudomonas isolated from rhizosphere
soils of different chickpea growing area in Punjab state of India, has a potential
to suppress chickpea wilt under in vitro conditions (Paul et al.,
1999; Kaur, 2003). The objective of the present study was to determine the biocontrol
efficacy of these selected Indian isolates and three other antagonistic isolates
from France against chickpea wilt in growth chambers and in microplots when
applied singly as well as in combination.
MATERIALS AND METHODS
Isolations of F. oxysporum and fluorescent Pseudomonas:
The isolates of F. oxysporum and fluorescent Pseudomonas were
collected from the rhizosphere soil of chickpea plant growing in different area
in Punjab state of India. Komadas medium (Komada, 1975) was used as an
agar-based selective medium to isolate F. oxysporum from soil samples.
The isolates of fluorescent Pseudomonas were isolated on King' B medium
(King et al., 1954) and purified by streaking.
Selection of pathogenic and nonpathogenic isolates of F. oxysporum: All the isolates of F. oxysporum were screened on three weeks old seedlings of chickpea for their pathogenicity test using rapid test technique (Roberts and Kraft, 1971). Isolates showing maximum wilting with in shortest duration of time were recognized as potential pathogenic, while those showing no wilting was selected as nonpathogenic. Finally the isolate Fo52 was selected as nonpathogenic and one isolate F. oxysporum f. sp. ciceri (Foc) was identified as pathogenic isolate (Kaur et al., 2003).
Selection of antagonistic isolates of fluorescent Pseudomonas: In vitro evaluation of fluorescent Pseudomonas isolates was conducted using dual culture technique on PDA with Foc (Kaur et al., 2003). The bacterial isolates showing maximum zone of inhibition was selected for further studies.
The isolates Fo47 and Fo47b10 of nonpathogenic Fusarium and C7R12 of fluorescent Pseudomonas were procured from INRA, Dijon France, which were used as biocontrol agents against wilt pathogens in the France. These were isolated from Fusarium suppressive soil from Chateaurenard region of France (Alabouvette, 1986).
Evaluation of biocontrol efficacy of antagonists: To evaluate biocontrol efficacy of antagonist, soil and seed inoculations were done to conduct bioassay in growth chambers and only seed inoculation was done for microplot studies.
Bioassay in growth chambers
Soil inoculation: The soil was sterilized twice at 120°C in the autoclave for 30 min at 24 h interval. Each pot was filled up with l00 g-sterilized soils. The pots were inoculated with the wilt pathogen, Foc at the inoculum concentration of 1x103 cfu g-1 soil. For soil inoculation of the biocontrol agents, the charcoal based formulation of the nonpathogenic Fusarium Fo47, Fo47b10 and Fo52 at the rate of 1x106 cfu g-1 soil and of the fluorescent Pseudomonas C7R12 and Pf5 at the rate of 1x108 cfu g-1 soil were added separately two days after inoculation of the pathogen. Further, two days later the surface disinfected 5 seeds of wilt susceptible variety JG-62 were planted in pots. All the treatments were replicated five times. A constant temperature of 28°C was maintained for duration of study. The irrigation was given as required to maintain optimum moisture level. The wilt incidence was recorded at regular intervals.
Seed inoculation: For seed application of the biocontrol agents, the surface sterilized seeds were treated with charcoal based formulation (Kaur et al., 2003) of fungal and bacterial antagonists containing the inoculum at the rate of 1x106 and 1x108 cfu g-1, respectively. The carboxy methyl cellulose (CMC) was used as a sticker at the rate of 1% in the formulation. In case of combined treatments of nonpathogenic Fusarium and fluorescent Pseudomonas, the seeds were first treated with fluorescent Pseudomonas inoculum under suspensions at the rate of 1x108 cfu mL-1 and 24 h later seeds were inoculated with charcoal based formulation of fungal antagonists. After the treatments, the seeds were planted in pots containing 100 g soil and were inoculated with Foc at the rate of 1x103 cfu g-1 soil. The sowing was done as described for soil treatments. All the treatments were replicated five times. The irrigation was given as per requirements. The wilt incidence was recorded at regular intervals.
Bioassay in microplots: The microplots each of size 2x1.5 m size were prepared in experimental area of Department of Plant Pathology, Punjab Agricultural University, Ludhiana. The 60 mL of Foc inoculum at 1x106 cfu mL-1 were inoculated in each plot 5 days of sowing. The chickpea seeds treated with charcoal based formulation of three nonpathogenic Fusarium and two fluorescent Pseudomonas isolates were sown after five days of inoculation of pathogen. Microplots consisted of 5 rows. Initially 25 seeds were sown in each row, however after germination only 15 seedlings were maintained for further observations. All the treatments were replicated three times.
Statistical analysis: Data from wilt incidence was subjected to ANOVA using SAS (SAS Institute Inc.) and treatments means were separated by Tukeys HSD test at p = 0.05. Before analysis the data was transformed by √x+1 transformation to equalize the error variances prior to analysis of variance. Untransformed data has been shown in the tables.
RESULTS AND DISCUSSION
The treatments of nonpathogenic Fusarium and fluorescent Pseudomonas
singly as well in combination were highly effective to reduce the wilting in
chickpea (Table 1). Only 15-30 and 20-35% plants showed wilting
after 30 and 60 days of sowing respectively when isolates of nonpathogenic Fusarium
were applied singly as soil treatment, where as in control 100% plants were
wilted after 30 days. Soil treatment with isolate C7R12 of fluorescent Pseudomonas
protected plants from wilting up to 30 days but after 60 days of sowing 40%
plants were wilted. Similarly the soil treatment with isolate Pf5 produced 15
and 50% wilted plants after 30 and 60 days of sowing. Soil treatment with combination
of non- pathogenic Fusarium with fluorescent Pseudomonas gave
better protection against F. oxysporum f. sp. ciceri. The combination
of Fo52+C7R12 was the best in controlling F. oxysporum f. sp. ciceri
infection, as there was no wilting after 30 days of sowing and only 10% wilting
recorded after 60 days of sowing.
All the 5 antagonists when applied as seed treatment singly or in combination gave significant protection against wilt pathogen (Table 1). There were 15-20 and 25% plants wilted after 45 days of sowing in seed treatments in nonpathogenic Fusarium and fluorescent Pseudomonas respectively as compared to 95% wilting in control.
Germination of seeds and wilting of chickpea plant reduced significantly due to application of nonpathogenic Fusarium and fluorescent Pseudomonas singly or in combination at various stage of plant growth in microplot studies (Table 2). The 68-69, 64-65 and 68-74% seed germination was recorded due to treatments of nonpathogenic Fusarium, fluorescent Pseudomonas and combination of both antagonists respectively as compared to 57% germination in control. The combined treatment of Fo52+C7R12 gave maximum, 74% germination of seeds. Among the nonpathogenic Fusarium isolates, the treatment with Fo47b10 gave complete protection up to 30 day of sowing. However, 8 and 4 wilted plants were observed in plots treated with Fo47 and Fo47b10, respectively. The treatment of fluorescent Pseudomonas also gave better protection and only 38 and 58% plants showed wilt symptoms when treated with Pf5 and C7R12, respectively. The combination of Fusarium and Pseudomonas treatments was found better than the single application of either the antagonist. Only 0-4% wilting observed in all the 6 combined treatments after 30 days of sowing as compared to 10% wilting in control (Table 2).
Efficiency of nonpathogenic Fusarium and fluorescent Pseudomonas against fusarial wilt of chickpea had already been described in different parts of world (Alabouvette et al., 1998; Paul et al., 1999). Current studies were conducted to know about the possibility to use nonpathogenic F. oxysporum as seed and soil treatment under Indian conditions. The Indian strain of nonpathogenic Fusarium isolated from rhizosphere of
||Effect of seed and soil inoculation of nonpathogenic Fusarium
oxysporum and fluorescent Pseudomonas singly as well as in combination
on the incidence of chickpea wilt in growth chambers at 26°C
|*Days after sowing, Nonpathogenic F. oxysporum- Fo52,
Fluorescent Pseudomonas-Pf5 and C7R 12, Means±SEM within a column
followed by different lower case letter(s) are significantly different (p =
0.05) using Tukey HSD test chickpea also reduced the fusarial wilt in chickpea
(Paul et al., 1999; Kaur, 2003).
||Fect of seed inoculation of nonpathogenic Fusariumo xysporum
and fluorescent Pseudomonas singly as well as in combination on the
incidence of chickpea wilt in field conditions
|*Days after sowing, Nonpathogenic F. oxysporum-Fo52,
Fo47, Fo47b10, Fluorescent Pseudomonas-Pf5 and C7R12, Means within
a column followed by different lower case letter(s) are significantly different
(p = 0.05) using Tukey HSD test
Fluorescent Pseudomonas strain, Pf5 was effective to reduce the incidence
of several soil borne pathogen including, pathogenic F. oxysporum (Kaur
et al., 2003; Sandhu, 2001). Individually the efficacy of nonpathogenic
Fusarium and fluorescent Pseudomonas varied much from one host
system to other. These results were very interesting since seed application
is only economical way to apply biocontrol agent in open fields. These results
obtained under field conditions in an artificially infested soil show that these
biocontrol agents might be effective in growers fields. Development of
biological control products based on these strains needs technological research
to study the best formulation to ensure success of the control.