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K. VENKATA RAMANAMMA, B.V.BHASKARA REDDY, R. SARADA JAYALAKSHMI, K.V.HARI PRASAD, G. MOAHN NAIDU AND V. JAYALSKHMI.
Dept. Of Plant Pathology, S.V Agricultural College, ANGRAU, Tirupati – 517 502, Chittoor Dt., Andhra Pradesh, India.
A total of 12 fluorescent Pseudomonas isolates collected from rhizosphere soils of healthy chickpea plants from six districts of Andhra Pradesh were evaluated against chickpea Fusarium wilt pathogen i.e., Fusarium oxysporium f.sp. ciceris by dual culture technique. Significant differences in inhibition percentage were observed among the treatments. Maximum per cent inhibition was observed with CRP-6 isolate (58.32%) followed by CRP-8 (56.90%) and minimum per cent inhibition of 4.57 was observed in CRP-10 isolate.
Chickpea, fluorescent Pseudomonas, dual culture, Fusarium.
Chickpea (Cicer arietinum L.) is an important pulse crop of India, belonging to leguminasae family and commonly known as Bengal gram. India occupies world’s 70 per cent of chickpea cropped area and 67 per cent production and Andhra Pradesh contributes total 7 per cent of production in the country approximately. In A.P, it is grown in an area of 3.42 lakh ha with a production of 3.91 lakh tonnes accounting for productivity of 1143 Kg ha-1 (Anonymous, 2017).
Among the diseases, Fusarium wilt caused by Fusarium oxysporum f.sp.ciceris (Padwick) Matuo & K.Sato (FOC) is highly destructive and worldwide in occurrence (Nene et al., 1989). The disease appears at almost all stages of plant growth. It survives in the soil for many years and is also seed borne in nature. Rhizosphere bacteria such as Pseudomonas have proved to be effective biocontrol agents against soil borne diseases of many crop plants. In this experiment, isolation of Pseudomonas and their efficacy was tested against the wilt pathogen i.e., Fusarium oxysporum f.sp. ciceris.
The pathogen Fusarium oxysporum f.sp.ciceris (Foc) was isolated from the wilt affected parts of chickpea plant and its pathogenecity was proved on sus-ceptible chickpea cultivar i.e., JG-62. The pathogen Fusarium oxysporum f.sp.ciceris (Foc) was isolated from the wilt affected parts of chickpea plant and its pathogenicity was proved on susceptible chickpea culti-var i.e., JG-62. For isolation of Pseudomonas, serial di-lution method proposed by Johnson and Curl (1972) was followed. For isolation, composite soil samples were col-lected from rhizosphere of healthy plants of chickpea. The soil was dried under shade and then used for serial dilu-tion. For isolation of Pseudomonas 10-3 to 10-6 dilutions were prepared and 0.1 ml of the required dilution was spread onto sterilized Petri plates containing cooled Kings B medium (King et al., 1954). After solidification they were kept in an incubator at 28±2oC and observed at fre-quent intervals for the development of colonies. Three Petri plates were maintained for each dilution. After 48 hours of incubation from the dilution plates, transparent, smooth margined bacterial colonies were selected and exposed to UV light in UV chamber at 365 nm for few seconds and based on fluorescens they were identified as fluorescent Pseudomonads. Then these colonies of Pseudomonas were transferred onto fresh Nutrient agar/ Kings-B medium by streak plate method (Dilip Kumar and Dube, 1992) and pure cultures were obtained. All purified isolates were found Gram-negative and rod shaped upon Gram staining.
After confirmation of fluorescent nature of these isolates, they were tested against the pathogen by dual culture method. A 5 mm disc of pathogen (one week old culture) was placed at the centre of PDA plate. Antago-nistic bacteria were streaked individually on both sides of the pathogen at 2.5 cm distance leaving 2.0 cm from pe-riphery. Plates inoculated with pathogen alone served as control. Three replicates were maintained for each bac-terial bioagent and incubated at 28 ± 2oC. Observations were recorded after one week to see the effect of an-tagonism on pathogen by bioagents. Per cent inhibition of mycelial growth of test pathogen over control was cal-culated by using the formula.
C – T
Percent inhibition (I) = ——— X 100
C
C = Radial growth (mm) of pathogen in control plate.
T = Radial growth (mm) of the pathogen in treatment plate.
Two potential Pseudomonas isolates from the above experiment was selected and qualitative HCN production was estimated by following Bakers and Schippers (1987) method.
A total of 12 fluorescent Pseudomonas isolates were isolated from chickpea rhizosphere of healthy plants on Kings B medium. Out of suspected 20 isolates, 12 isolates were identified and confirmed as fluorescent Pseudomonads based on fluorescens nature and these isolates were numbered from CRP 1 to CRP 12 (Table 1, Fig.1). Gram staining of these 12 fluorescent Pseudomo-nas isolates exhibited Gram negative reaction. This is in accordance with King et al. (1954) who isolated Pseudomonas spp. from rhizosphere soil of chickpea plants by using King’s B medium. Sharma (2012) iso-lated 23 Pseudomonas isolates from rhizosphere and rhizoplane of different crops including chickpea and iden-tified based on their growth on Pseudomonas selective (fluorescein) agar medium and Gram staining.
Dual culture test results indicated that among 12 Pseudomonas isolates evaluated against the pathogen the isolate CRP-6 recorded highest inhibition percentage of 58.32 (Table 2) followed by the isolate CRP-8, which recorded inhibition percentage of 56.90 (on par with
each other). The isolates CRP-7, CRP-11 and CRP-9 – 106
recorded inhibition percentage of 43.21, 41.10 and 41.00 respectively and were on par with each other. The lowest percentage of inhibition was recorded with the isolate CRP-10 (4.57).
The experimental results were in agreement with Kandoliya and Vakharia (2013) who isolated ten Pseudomonas fluorescens isolates from chickpea rhizosphere and evaluated against Foc in dual culture and noted highest per cent inhibition of 83.5 with Pf-3. In similar experiment, Manjunatha et al. (2012) evaluated two already identified potential Pseudomonas isolates viz., Pf-4 and Pf-6 against F. udum and recorded more inhibition in Pf-4 than Pf-6.
Generally, the biocontrol quality of antagonistic bacteria involves either competition or production of metabolites such as HCN, siderophores, an-tibiotics or extracellular enzymes etc., that acts antagonistically towards the plant pathogens (Sang et al., 2006).
In the present study clear inhibition zones were observed in dual culture of pathogen with Pseudomonas (CRP-6, CRP-8). Highest inhibition zone of 6 mm was observed in the isolate CRP-8 followed by CRP-6 which has an inhibition zone of 4mm. It might be due to the antifungal substances and/or cell wall degrading enzymes released by the bacteria into the culture medium (Fatima et al., 2009). The area of inhibition zone was taken as a measure of antagonistic potential of the isolate. Pseudomonas fluorescens inhibits the development of mycelium much more with volatile compounds.
These two potential isolates i.e. CRP-6 and CRP-8 also produced HCN which was confirmed by the change in the colour of the filter paper form yellow to reddish brown (+++). HCN effectively blocks the cytochrome oxidase pathway and also inhibits the transport of electrons thereby disrupting energy supply to the cell leading to the death of the organism (Manwar et al., 2011). Manjunatha et al. (2012) reported that two strains of Pseudomonas Pf-4 and Pf-6 strains produced antibiotic compounds such as phenazine, HCN and salicylic acid that showed antagonistic activity towards F.udum.
In the present experiment, in vitro studies indicated that two fluorescent Pseudomonas isolates i.e. CRP-6 and CRP-8, have shown highest inhibition activity against Fusarium oxysporum f.sp. ciceris.
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