The Effects of Colpoda inflate Predation on Pseudomonas syringae in Bean Phyllosphere Amy Ip Abstract Pseudomonas syringae has caused brown spot disease and frost damage to a variety of commercial crops such as snap bean Previous studies have indicated that abundant populations of P syringae cause plants to be more susceptible to pathogenic harm This exploratory study considered how protist predation may increase the fitness of bean leaves and thus the whole plant The chosen predator species Colpoda inflata a common soil ciliate is known to be capable of surviving on the phyllosphere Bean leaves were inoculated with P syringae and with or without C inflata The effects of predation were measured by enumerating bacteria and protist populations P syringae populations were counted with spiral plating Results are showed that P syringae populations are maintained at steady levels in the presence of protist predation However visual health and brown spot counts indicate protist predation does not eliminate brown spot disease from bean plants While protozoa presence may not reduce disease risk this study suggests that microbial communities containing protist and bacteria can coexist in the phyllopshere Further studies are needed to understand the mechanisms of epiphytic microbial communities Introduction The phyllosphere or the leaf environment of plants sustains microbial communities some of which may be beneficial or detrimental to a plant s overall fitness Ruinen 1961 Hirano and Upper 2000 While many microbes may inhabit the phyllosphere Pseudomonas syringae a well studied bacteria plant pathogen is known to contribute to brown spot disease and frost damage of leaves Gross and Devay 1977 Lindow et al 1978 Lindemann et al 1984 Hirano and Upper 2000 Studies have suggested large epiphytic populations of P syringae species plant diseases Lindemann et al 1984 Rouse et al 1985 If these large bacterial populations can be reduced the severity of brown spot disease may also be alleviated Protozoa a natural predator of bacteria have been found to survive the daily flux of temperatures on leaf surfaces Bamforth 1971 Tanner 1975 The protozoan predation of bacteria has been found to reduce bacterial numbers in freshwater environments The study carried out by Drake and Tsuchiya 1978 suggested that Colpoda steinii helped decrease populations of Escherichia coli Cox et al 1999 demonstrated the potential of using protozoan predation as a means to reduce biomass accumulations on filters Like filters organisms generally adhere to leaf environments unless the environment is disturbed A protist likely to be found on leaf surfaces is Colpoda inflata a soil ciliate Hausmann and H lsmann 1996 Though these studies opened further discussion on bacteria protozoa interactions bacteria and protozoa inhabiting on a leaf and their potential roles in plant pathology has not been documented before This experimental study scratches the surface of protozoan and bacteria interactions in the phyllosphere by studying the effects of a soil ciliate Colpoda inflata on epiphytic populations of P syringae on healthy bean leaves The hypothesis that Colpoda inflata would significantly reduce populations of P syringae was investigated Methods C inflata was grown in cerophyll medium Sonneborn s Paramecium prepared similar to the one used in Pratt et al s study 1997 Cerophyll leaves were boiled in 1 L of deionized distilled water for 5 minutes and filtered through 1 Whatman filter paper Enterobacter aerogenes ATTC 13048 was isolated from the initial protozoan culture and maintained on Luria s agar plates to use as feed bacteria in subcultures Subcultures were carried out 7 10 days for about 2 months In preparation for inoculating on bean leaves about 100 mL of C inflata was centrifuged for approximately 2 minutes at 2000 Gs The supernatant was removed and about 80 mL of fresh grass medium was added for every 20 mL of protozoan pellet Of the five or eight treatment samples 20 ul of three leaf samples were scanned three times each with a light microscope at 16X for C inflata presence P syringae B728a was grown on King s medium B with rifampincine benlate and natamycin The bacteria was suspended in KPO4 buffer whose concentration was determined with Spec 20 Appropriate amount of grass medium was added to give a concentration of 106 cells mL Bean plants housed in a mist chamber were sprayed with either only P syringae or P syringae and C inflata Individual leaves were carefully placed in test tubes with deionized sterile water and placed in a sonicater Branson 8200 for 7 minutes P syringae counts were determined by spiral plating on the same King s medium B plates as above For each of the two variation of treatment 5 leaf samples were taken for Day 0 and 8 leaf samples subsequently for Day 1 2 and 3 Results Day 1 and 3 were found to be significant by two tailed t test p 0 05 Day 0 was not found significant as expected Populations were not significantly different on Day 2 Brown spot counts were taken five days after Day 3 to determine the health of plants Counts were not significantly different also using two tailed t test p 0 05 Effects of C inflata Predation on P syringae Populations 9 000 8 000 7 000 log cfu g 6 000 5 000 Control 4 000 Treatment 3 000 2 000 1 000 0 000 0 1 2 3 day Figure 1 Control refers to plants sprayed only with P syringae Bean plants subjected to treatment were sprayed with both P syringae and C inflata Brown Spot Counts with or without C inflata 30 25 of spots 20 15 10 5 0 Control Treatment Figure 2 Control refers to plants sprayed only with P syringae Bean plants subjected to treatment were sprayed with both P syringae and C inflata Discussion Since samples of Day 0 were taken immediately after inoculation the difference in populations should be explained by chance as the results show Figure 1 The inoculums of P syringae with and without C inflata were made on Day 0 so the protozoa did not have time to change bacterial numbers By Day 1 on the control plants it appeared P syringae populations exploded without predation keeping them in check Most plates of Day 1 were undistinguished because of high growth so populations were estimated to be 108 cfu g The 100 fold increase from Day 0 to Day 1 in the control samples is at best a general estimate The subsequent 10 fold decrease suggested that the carrying capacity of the leaves decreased so many bacteria could not survive Nutrient levels can dictate the growth of
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