Abstract: Twenty isolates of Pseudomonas sp., were tested for the control of soilborne root-infecting fungi like Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani under laboratory and greenhouse conditions. In dual culture plate assay, strain 78 inhibited all the test fungi. Other Pseudomonas sp., failed to check the radial growth of M. phaseolina whereas isolates 51 and 82 significantly inhibited radial growth of F. solani and R. solani. Three isolates of Pseudomonas sp., (51, 78 and 82) when used as seed dressing or as soil drench showed substantial reduction in root-rot infection caused by M. phaseolina, F. solani and R. solani in mungbean. Strain 78 was found to be most effective in the control of root-infecting fungi. Of the root-infecting fungi, R. solani was most susceptible to Pseudomonas sp.
Introduction
Several soilborne pathogens cause root-rot and root-knot diseases in mungbean {Vigna radiata (L.) Wilczek)} and may seriously limit production (Siddiqui et al., 1999). Of these, Fusarium solani (Mart.) Appal and Wollenw. Emend Snyd. and Hans., Macrophomina phaseolina (Tarsi) Gold, Rhizoctonia solani Kuhn are reported to cause serious problems in mungbean production (Ehteshamul-Haque and Ghaffar, 1994). Since many crops have little or no resistance to many diseases, use of microorganisms in the biological control of disease is an alternate method of plant disease control (Lumsden and Lewis, 1989).
Research on biological control agents has emphasized free-living, plant-associated, non-pathogenic bacteria present in the rhizosphere and rhizoplane. Of the various rhizosphere bacteria, Fluorescent pseudomonads (Pseudomonas fluorescens, P. putida, P. aeruginosa and some closely related species) are common inhabitants of the rhizosphere and contribute to the control of many soilborne plant diseases caused by fungi (Alabouvette et al., 1993; Buchenauer, 1998). In this paper we discuss some experiments on the effectiveness of Pseudomonas bacteria in the control of root-infecting fungi such as Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani.
Materials and Methods
Microorganisms used: Twenty strains/isolates of Pseudomonas sp., were obtained from the culture collection of the Department of Genetics, University of Karachi. The bacteria were maintained on King's B medium for 5 days at 25±2°C and used in the present study. Root-infecting fungi viz., M. phaseolina, F. solani and R. solani were isolated from the infected roots of mungbean and maintained on potato dextrose agar (PDA) at 25±2°C for one week.
In vitro studies: Using technique as suggested by Drapeau et al. (1973), the strains/isolates of bacteria were streaked on one side of the Petri dish containing Czapek's Dox Agar medium, pH 7.2 whereas on the other side a 5-mm diem., disc of root-infecting test fungus was inoculated. The dishes were incubated at 25±2°C and zone of inhibition (if any) was recorded after one week.
Greenhouse studies: Of the twenty isolates of Pseudomonas sp., used in the present study, only three showed promising results in the inhibition of radial growth of root-infecting fungi and were selected for their use as seed dressing and soil drench in the control of root-infecting fungi on mungbean under greenhouse conditions. Soil used in the present study was a sandy-loam (sand:silt: clay, 70:19:11), pH 8.1 with moisture holding capacity of 40% obtained from the experimental field of the Department of Botany, University of Karachi. The soil had a natural population of 4 to 11 sclerotia g–1 of soil of M. phaseolina as assessed by wet sieving and dilution technique (Sheikh and Ghaffar, 1975); 7% colonization of R. solani on sorghum seeds used as baits (Wilhelm, 1955) and 2800 CFU g–1 of soil of Fusarium solani as assessed by soil dilution technique (Nash and Snyder, 1962). The soil was transferred in 8-cm diam., plastic pots at 350 g per pot.
For seed dressing, mungbean seeds after surface sterilization in 1% Ca(OCI2 for 3 mins., were rinsed several times with tap water and treated with aqueous cell suspension of the bacteria using 1% gum arabic as sticker giving a population of 2.0-2.4×106 CFU per seed. After seed treatment eight seeds were sown in each pot and after germination only four seedlings were maintained per pot. Seed treated with sterile distilled water served as controls. For soil treatment, after removing the soil to a depth of 2.5 cm a 25 ml aqueous cell suspension containing 3.0×106 CFU ml–1 was drenched in each pot. Eight mungbean seeds were then sown in each pot and surface was covered with soil. After germination four seedlings were kept in each pot. Soil drenched with sterile distilled water was used as a control. There were three replicates of each treatment and pots were kept randomized on greenhouse bench of the Soilborne Diseases Research Laboratory, Department of Botany, University of Karachi. The temperature of the upper soil surface ranged between 25-30°C throughout the experiments.
The plants were uprooted carefully after 45 days growth. To determine the incidence of root-infecting fungi, 1 cm long root pieces were surface sterilized with 1% Ca(OCI)2 and transferred onto PDA plates supplemented with penicillin (100, 000 units/I.) and streptomycin sulfate (0.2 g/I.) 5 pieces/plate. The plates were incubated at room temperature and after one week infection on roots by fungi was recorded using the following formula:
Results
In dual culture plate assay, isolates of Pseudomonas showed variable activity against different root-infecting fungi. Strain 78 of Pseudomonas sp., inhibited all the test fungi producing a zone of 8 and 5-mm respectively against M. phaseolina and F. solani whereas colonies of bacteria and R. solani met each other with no further growth recorded of either organism. Similarly, strain 82 produced a zone of 16 and 10 mm against F. solani and R. solani respectively, whereas strain 51 produced a 20 and 10 mm zone of inhibition respectively, against F. solani and R. solani. Most of the bacterial isolates used in this study were failed to inhibit M. phaseolina (Table 1).
| Table 1: | In vitro inhibition of Macrophomina phaseolina, Fusarium solani and Rhiroctonia solani by different strains/isolates of Pseudomonas spp. |
| *A =Colonies of both organisms met each other with no further growth of either organism observed. B =Colonies of the pathogen overgrew on the bacterium. C =A zone of inhibition was initially produced but later on fungus overgrew on the bacterial growth. |
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Under greenhouse conditions, Pseudomonas isolates used as seed dressing or as soil drench although reduced root infections caused by M. phaseolina and F. solani but their effects were statistically non-significant. Strain 78 of Pseudomonas sp., used as seed dressing (>46 %) or as soil drench (>39%) showed greatest reduction in F. solani infection. R. solani infection was significantly (p<0.05) suppressed following seed dressing with Pseudomonas sp., with no significant effects when used as soil drench. Strain 51 and 78 used as seed dressing (>75%) or as soil drench (>66%) resulted in the maximum reduction in R. solani infection (Fig. 1).
Discussion
In dual culture plate assay Pseudomonas sp., inhibited radial growth of root-infecting fungi viz., M. phaseolinha, F. solani and R. solani producing zones of inhibition. It interesting to note that different strains of Pseudomon showed variability against different root-infecting funge. Presumably like rhizobia (Chao, 1990), the antagonist ability of P. aeruginosa also varies with the strains used.
| Fig. 1: | Effect of seed dressing or soil drench of different strains/isolates of Pseudomonas bacteria in the control of Macrophomina phasolina, Fusarium solani and Rhizoctonia solani in mungbean A = Control, B = Strain 82, C = Strain 51, D = Strain 78 |
In the present study, Pseudomonas aeruginosa and Pseudomonas sp., either used as seed dressing or as soil drench significantly prevented the infection of M. phaseolina, F. solani and R. solani. The bacteria belonging to the fluorescent pseudomonas eg., Pseudomonas fluorescens and P. putida which colonize roots of a wide range of crop plants are reported to be antagonistic to soilborne plant pathogens like Rhizoctonia solani, Pythium ultimum, P. debaryanum and P. aphanidermatum (Suslow and Schroth, 1982). Cultures of P. fluorescens used for the seed treatment of wheat showed control of take all disease of wheat (Weller and Cook, 1983). Banana suckers when treated with P. fluorescens also reduced the severity of wilt and internal discoloration on banana (Sivamani and Gnanamanickam, 1988). P. fluorescens reduced the infection of Sclerotiurn rolfsii on peanut (Ganesan and Gnanamanickam, 1987). Fluorescent Pseudomonas has also been reported to reduce Fusarium wilt in carnation (Yuen et al., 1985).
In the present study, it is interesting to note that R. solani was found comparatively susceptible to Pseudomonas sp. There are reports where strains of Pseudomonas fluorescens and P. putida carrying the chiA gene coding for chytinolytic enzyme suppressed disease caused by the plant pathogenic fungi Rhizoctonia solani and Sclerotium rolfsii (Koby et al., 1994). Most of the Pseudomonas species are known to survive for a shorter period of time in the rhizosphere after introduction in the soil. Downing and Thomson (2000) in their bean model system demonstrated that P. fluorescens Rifl was only required for a brief period as R. solani, the main cause of damping-off disease, rapidly colonizes seedlings and reaches a peak within 2 to 4 days (Sneh et al., 1966). It is therefore suggested that effective biological control using Pseudomonas bacterium can be attained in those crop-fungus combinations where yield loss can be prevented by reducing fungal colonization within the first week after germination.
Acknowledgment
The present work was carried out under the research grant of University Grants Commission, Pakistan which is sincerely acknowledged.