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SCREENING BACTERIA FOR ROOT COLONIZING ABILITY BY A RAPID METHOD I.
Department of Plant Pathology. University of Arizona. Tucson. AZ 85721, U.S.A. 5 June 1990)
Summary-A method is described for a rapid screening of a large number of bacterial isolates for their root colonizing ability based on direct microscopic observation of roots grown in water agar. Root colonizing bacteria could be seen swarming around roots and forming highly visible bright halos within 7-12 h after bacterial suspensionswere placed near the root tips. To determine the relative value of the direct microscopic method, 16 randomly selected bacteria1 isoIates were screened for their root colonizing ability by the direct observation of roots of cucumber and tomato grown in agar and by the standard dilution plating method using roots grown in soil. The rankings of the isolates for root colonizing ability obtained by the two methods were highly corretated. In 32 plant-bacterium combinations tested. the outcome of only two combinations were different in the two methods. One isolate which colonized cucumber in water agar and another isolate which colonized tomato roots in water agar did not colonize roots of these plants in the soil. Results show that the direct microscopic method can be used for a rapid qualitative screening of bacteria for their ability to colonize roots of certain plants.
Non-nitrogen fixing thizobacteria that arc capable of promoting plant growth or protecting plants against
pathogens have been the subject of numerous studies (Burr et al.. 1978; Schcr and Baker, 1980; Schroth and Hancock, 1981; Suslow and Schroth, 1982; Cook and Baker, 1983; Kloepper, 1983; Loper er al., 1984; Bahm
1987; Schcr et a/.,
Weller, 1988). Results of these studies, taken together, indicate that root colonizing ability (the ability to build populations on or around roots) is an essential prerequisite for the success of rhizobacteria in serving useful functions. This should be expected because root colonizing ability (RCA) which is highly variable among rhizobactcrial isolates (Bennett and Lynch, 1981; Suslow and Schroth. 1982; Lynch and Clark, 1984; Scher ef al., 1988), is a reflection of the ability of these rhizobacteria to compete for ecological niches in the highly competitive rhizosphere. The current methods of evaluating RCA involve the placing of test isolates on or around roots, reisolating them by washing or triturating roots after a few days and determining bacterial cells by dilution plating methods (Bowen and Rovira. 1976; Olsen and Bakkcn, 1987). Population size values obtained using dilution plating methods are highly variable (Burr ef al., 1978; Olsen and Bakken, 1987) necessitating numerous repetitions. Moreover, the methods are time consuming and, therefore, are not practical for screening large numbers of bacterial isolates for RCA. A method is described for the rapid qualitative screening of large numbers of bacterial isolates using a unique direct microscopic technique. MATERIALS AND METHODS
Isolates used in this study were from the rhizosphere and rhizoplanc of roots of cotton, tomato,
cucumber and alfalfa plants grown in the field. Roots first were shaken gently to rcmovc loose soil particles and ca 200 mg of root sections wcrc placed in 300 ~1 of 0.1 M phosphate buffer. pH 7.0 in a 2.5 ml centrifuge tube. Tube contents were shaken on a vortex shaker platform for 20 min at high speed and diffcrent dilutions of the root washings were plated on King’s Medium B (KB) agar (King et al.. 1954). Discrete bacterial colonies were sclcctcd after 24 h at 25-27’C and their purity was tcstcd by at least three cycles of streaking and reisolation from single colonies on KB plates. Sixteen isolates (3 Grampositive and 13 Gram-negative) in the total recovered population from all plants were selected for RCA tests. Rifampicin- and nalidixic acid-resistant mutants of all the selected isolates were obtained on KB agar supplemented with IOOpg ml-’ of each antibiotic. The stability of the antibiotic resistance trait was confirmed by three cycles of streaking and reisolation from single colonies on KB without antibiotics and then restreaking on antibiotic supplemented KB. The selected isolates were characterized using standard biochemical tests (Misaghi and Grogan. 1969) and API 20E and API 50 diagnostic tests (API Analytab Products, Plainview, N.Y.).
Bacterial isolates were screened for their RCA by a direct microscopic method and by dilution plating method and results were compared. For the direct microscopic method, seeds of cucumber, cv. Straight 8, and tomato, cv, Earlypak, were germinated in 86.mm-dia. plastic Petri plates containing 25 ml of 1.2% agar. Eight to IO seeds of cucumber or tomatoes were embedded in the agar in a straight line, co 3 cm from the center of the Petri plates. Seeds were not surface sterilized prior to sowing. Plates were stored on edge at 25-27°C in the dark to allow the roots to grow down through the agar for co 3 cm
Fig. I. A diagram of 3-day-old tomato seedlings grown in a Petri plate containing water agar, showing seeds (S), roots (R) and points of inoculation (PI). See the text for detail. from the seed line (Fig. I). About 2 ~1 of a bacterial suspension (I x IO’cfu ml-‘) from a 24-h-old KB
culture of each of the 16 antibiotic-resistant were introduced
into agar at cu 2 mm below the tips
of roots growing near the bottom of a Petri plate by inserting the tip of a sterile Pasteur pipette filled with cu 10~1 of the bacterial suspension into the agar. Plates were again stored on edge at 25-27’C in the dark to allow the root to grow through the sites of bacterial deposit. To visualize root colonization, a Petri plate was placed inverted on a stage of a compound microscope, I2 h after bacterial deposit. and the roots wcrc obscrvcd using a IOx objective Icns. Although a few hactcrial cells could occasionally bc seen swarming around some roots, visualization was enhanced greatly and visibility was greatly improved by the following adjustments: (I) the objcctivc lens platform was turned CCJ f IO dcgrccs so that the IOx objcctivc lens was slightly out of alignment; (2) the light intensity was increased to the maximum; (3) the substage condcnscr diaphragm was partially closed; (4) the root was brought back into the field; (5) the light intensity, the substage condenser diaphragm, and the degree of the misalignment of the objective lens were readjusted for maximum visibility
These adjustments improved the quality of the image markedly, making it possible to see bacterial cells very clearly as moving bright spots around roots. To determine if the percent of roots colonized by any one of the I6 isolates changes with time, roots were observed microscopically I2 h, I day and 3 days after bacterial deposit. However, since the percent remained unchanged, roots were examined only I2 h after bacterial deposit for the 5th and 6th repetitions. Each of the 16 bacterial isolates were screened for RCA by direct microscopic method six times and each time 12-18 seedings with straight roots were inoculated in two plates. Root colonization index was calculated for each isolate on tomato and on cucumber on a scale from zero (0% of seedlings were colonized) to 1.0 (lOO% of seedlings colonized). To screen bacterial isolates for their RCA by dilution plating method. 4-5-wk-old tomato and cucumber plants, grown in pasteurized soil in an environmental chamber with daily cycles of I2 h at 27-29°C plus light (372 Em-* s-l) and I2 h at 23-24°C plus dark, were gently removed from the soil and roots were washed under running water
for ca 30 min. Groups of four seedlings were transplanted each into a separate 4 x 25 cm Cone-tainer (Ray Leach Cone-tainer Nursery, Canby, Ore.), filled with a pasteurized soil : sand : peat moss (3 : I : I, v/v) mixture infested with one of the 16 antibiotic-labelled isolates. To infest soil with bacteria, rifampicin- and nalidixic acid-resistant isolates used in the study were grown on KB culture plates. supplemented with IOOpg rifampicin ml-’ and IOOpg nalidixic acid ml-’ for 24 h at 25-27°C. Colonies were suspended in sterile water and appropriate quantities of the bacterial suspension were mixed thoroughly into the soil mixture to provide I x IO’cfu g-l dry wt of soil. The moisture content of the soil mixture was adjusted to 20% which was equivalent to a matric potential of -8.5 kPa. based on a matric potential-moisture curve, determined by pressure membranes. To prevent evaporative loss of water, each Cone-tainer was placed inside a IO x 3Ocm plastic bag, the open end of the bag was pulled over the top of the Cone-tainer and around the seedling stem and was secured by a rubber band. Seedlings were returned to the same growth chamber environment and the population sizes of the introduced isolates in the rhizosphere were dctermincd I and 2 days after transplanting as follows: roots of four seedlings were removed from soil infcstcd with each of the 16 isolates and were gently cleaned of adhering soil particles. About 200 mg root samples from each plant. collected from an arca bctwecn I and 2cm from the crowns, were used to isolate antibiotic marked isolates. Isolation tcchniquc used was the same as those employed in rccovcring isolates from the soil except for the use of KB medium supplemented with rifampicin and nalidixic acid (IOOpg ml-’ each). Each of the I6 labcllcd isolate in each cxpcrimcnt was recovered from roots of four plants and the washings from each of the four roots was plated on two KB plates. The percent change in the population size of each isolate during the 24 h period (between I day and 2 days after transplanting) was calculated for each isolate. These tests were repeated four times each with four replicates and the results were subjected to statistical analyses. RESULTS
In the direct microscopic observation of the roots, a slight misalignment of the objective lens resulted in a marked increase in the quality of the image, making it possible to see clearly bacterial cells swarming around roots (Fig. 2). Dark field microscopy also improved image clarity over light microscopy. However, the images obtained by this method were not as clear as those obtained by misalignment of the objective lens. Phase-contrast microscopy was not practical because of the thickness of the agar. Roots grew into bacterial deposit sites in the agar within 4-6 h after inoculation. Some of the bacterial isolates colonized roots and could easily be seen swarming around roots as early as 7 h after inoculation and up to 5 days after inoculation. Swarming was particularly intense around root tips and root hairs, becoming less intense distal to root tips. This was probably due to the observed tendency of the bacteria to follow root tips as they advanced through
Screening bacteria for root colonizing ability the acar. Masses of motionless. presumably dead bactenal cells were occasionally observed around older roots. Similar observations have been made by Leben (1983). Swarming was not observed on non-inoculated roots. Bacterial isolates differed with respect to their ability to colonize roots of cucumber and tomato in water agar. Of the 16 isolates tested on tomatoes, five did not colonize any seedlings. one colonized 10% of the seedlings. one colonized 60% of the seedlings while nine colonized at least 80% of the seedlings (Table I). Similar patterns were observed with cucumber seedlings. Colonization was complete within I:! h after inoculation since the percent of roots colonized in each bacterium-plant combination (12-18 seedlings in two plates) was the same at IZh and at 3 days after inoculation. These results are based on six trials each with 12-18 seedlings. The percentage of the seedlings colonized by a single isolate was highly reproducible from one test to the other. A colonization index scale. ranging from 0 to I.0 was used to rank isolates for their RCA in the agnr test. Colonization indices of 0, 0.5 and 1.0 were assigned to isolates which respectively colonized 0. 50 and 100% of the seedlings in agar (Table I). In the soil the average number of cfu g- ’ fresh wt of tomato roots wcrc 2.6 x IO’ and 8.0 x IO’ one day and Z days after sccdlinps wcrc transplanted into the soil infcstcd with the test isolates. rcspcctivcly. The rcspcctivc figurss for cucumber wcrc 2.6 x IO’ and X.3 x IO’. The isolates aIs0 wcrc rank4 for their RCA in the soil basctl on 3 root colonization in&x scale ranging from - I .O (I -fold dccrcasc in population sizes in 24 h) to +7.0 (7-f&i incrcasc in population sires in 24 h, Tublc I). The pcrccntagcs iIt2 h;wxl on 16 values ohtaincd in four cxpcrimcnts each with four rcplicatcs. Although SD ol’population sires obtain4 by dilution plating method wcrc quite high, the patterns wcrc similar in all four rcpctitions. The ranking of isolates for RCA obtained by dilution plating method corrclatcd quite well with that obtained by direct microscopic method (Table I). In 32 plant-bacterium combinations tcstcd. the outcome of only two ditrcred bctwccn the two methods. Isolate 8 with a colonization index of 0.8 on cucumbsr roots in agar and isolutc I5 with the
Fig. 2. Bacterial swarm around the root of a S-day-old tomato seedling grown in 12% water agar. 14 h after P.wutfor~rorro.~j’uor~~c~~.r. isolate I4 was placed near the root tip.
Tabk I. Comparison of the results of screening of bactmal for
Tomato Isolate I
by ddution method
3 P. /luorescenr
4 P. muida
IO P. -jiuorescenr
16 P. ourida *Root
index of isolates in agar on a scale from zero (0%
of seedlings were colonized) Each
is an average
(P = 0.05)
roots were 2.6 x IO’ and X.0 x IO’ one
seedhnvs were transnlantcd
were 2.6 x 10’;md
16’ vaIues ohtaincd
in soil on a scale from
sizes in 24 h).
dav and IWO davs after
sizes in 24 h). to
I tesl. The
of seedlings colonired).
per g fresh wl of tomato infested
to I .O (100% value
inco the soil ti8ure5 for
8.3 x IO’. Each lipurc IS an aver;,gc trials
colonization index of 0.8 on tomato roots in agar did not colonize roots of these plants in the soil (Table I). The probability of obtaining this Icvcl of agrccmcnt, if the rankings by the two methods wcrc not corrcIatcd. is < I% according to the sign test (Lchmann, 1975). Antibiotic resistant mutants wcrc stablo and rctaincd their rcsistancc following their reisolation from roots. DtSCtJS..lON
The dilution plating method is being used almost universally for the asscssmcnt of bacterial population on roots of plants. Howcvcr, the method is relatively elaborate and time consuming, The prcscncc of a high degree of correlation bctwccn the dilution plating method and direct microscopic method described here shows that the latter method may be used for a rapid qualitative screening of a large number of bacterial isolates for RCA. The direct microscopic method is rclativcly quick, simple. and substantially less labour intensive and is perhaps the most practical method in studies where large numbers of bacteria1 isolates have to be scrcencd for root colonizing ability. A selcctcd few isolates can then be rated quantitatively for RCA using dilution plating tcchniquc. The direct microscopic method also can bc used for a quick dctormination of host sclcctivity of bacterial isolates and for studies of the biochemical as well as molecular aspects of root-bacterium intcractions. Another advantage of the direct microscopic method is that it is non-destructive which allows continuous observations of root-bacterium interactions and their dynamics. Moreover, it does not require special instrumentation.
Selectivity for root colonization has been reported (Azad er al., 1987). The fact that selectivity also was demonstrated in the agar tests suggests that this procedure may be useful in elucidating the nature of host selectiviy. Living bacterial cells have been observed around roots (Leben, 1983) and root cap cells (Hawes and Pueppke. 1987) under light microscope without any adjustment. Leben (1983) observed bacteria swimming in a film of water around the roots grown in water agar by direct microscopy. In the present study, some of the efficient root colonizing isolates could occasionally be seen around roots of both cucumber and tomato seedlings under the light microscope without the adjustments described earlier. However, the images were poor and often indiscernible compared to those obtained following the microscope adjustment. Both light microscopy and scanning electron microscopy have been used for qualitative and quantitative assessment of bacterial population sizes on roots. Rovira er al. (1974) have found a high degree of correlation between dilution plating method and direct microscopic observations for counting bacteria on stained root segments recovered from soil. Although their direct microscopic method is less time consuming than dilution plating method, it cannot distinguish between dead and living bacteria. The USCof scanning electron microscope (Rovira and Campbell. 1975) adds a new dimension to the study of root-bacterium associations and often complcmcnts data obtained by other studies. Qualitative asscssmcnt of the colonization ability of bacterial isolates also has been made on roots of plants grown in water agar by replica printing water agar slab onto bacteriological media (Lebcn. 1983) or by allowing roots to grow into nutrient media (Randhawa and Schaad, 1985). These methods, which are not in common use, are more time consuming than the direct microscopy method described hero. A root printing method has been developed for determining the in situ distribution patterns of bacteria on the rhizoplane of field-grown plants (Stanghellini and Rasmussen, 1989). REFERENCES Azad H. R.. Davis J. R.. Schnathorst W. C. and Kado C. I. (1987) Influence of verticillium wilt resistant and susceptible potato genotypes on populations of antagonistic thizosphere and rhizoplane bacteria and free nitrogen fixers. Applied Microbial Biotechnology 26. 99-104. Bahme J. B. and Schroth M. N. (1987) Spatial-temporal colonization patterns of a rhizobacterium on underground organs of potato. Phytopathology 77, 1093-I 100. Bennett R. A. and Lynch J. M. (1981) Bacterial growth and development in the rhizosphere of gnotobiotic cereal plants. Journal of General Microbiology 125, 94-102. Bowcn G. D. and Rovira A. D. (1976) Microbial coloniration of plant roots. Annual Review of Phytopathology 14, 121-144. Burr T. J., Schroth M. M. and Suslow T. V. (1978) Increased potato yields by treatment of seed pieces with specific isolates of PseudomonasJ%orescens and P. putida. Phytopathology 68, 1377-l 383.
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