control of tomato bacterial wilt without disinfection using a new functional polymer that captures...

Control of Tomato Bacterial Wilt without Disinfection

Using a New Functional Polymer That Captures Microbial

Cells Alive on the Surface and Is Highly Biodegradable

Nariyoshi KAWABATA,1;y Hitoshi KISHIMOTO,2 Takayuki ABE,1 Tomokatsu IKAWA,2

Katsumi YAMANAKA,2 Hisaya IKEUCHI,1 and Chizuko KAKIMOTO1

1Department of Materials Science, Faculty of Engineering, University of Shiga Prefecture,

Hassaka-cho, Hikone, Shiga 522-8533, Japan2Department of Chemistry, Faculty of Engineering and Design, Kyoto Institute of Technology,

Matsugasaki, Kyoto 606-8585, Japan

Received August 31, 2004; Accepted November 24, 2004

This report describes a green chemical method for

controlling soil-borne plant diseases without disinfection

using an equimolar copolymer of N-benzyl-4-vinylpyri-

dinium chloride with styrene (PBVP-co-ST) that cap-

tures microbial cells alive on the surface and is highly

biodegradable. Tomato bacterial wilt caused by Ralsto-

nia solanacearum was controlled by the addition of

sawdust coated with PBVP-co-ST prior to transplanta-

tion. This effected 87% reduction in appearance and

89% reduction in the index of symptom under appro-

priate conditions. The coated sawdust did not exhibit

bactericidal activity. The half-life of PBVP-co-ST was

5.6 d when treated with activated sludge in soil. The

disease control was explained in terms of reduction of

infectious contact between the roots of tomato and the

cells of R. solanacearum due to coagulation-like inter-

action between microbial cells and the coated sawdust,

in addition to capture of microbial cells by the coated

sawdust.

Key words: tomato bacterial wilt; Ralstonia solanacea-

rum; capture of microbial cells; poly(N-

benzyl-4-vinylpyridinium chloride-co-sty-

rene); half-life

Control of soil-borne plant diseases is a difficultsubject in agriculture. Conventional treatment processesinvolve disinfection using methyl bromide, chloropicrin,or other related chemicals as fungicides.1) But aninternational treaty decided on prohibition of methylbromide until 2005 because of its property of destroyingthe ozone layer.2) Hazardous chloropicrin is not desir-able. Under the conditions, an alternative method foreffective protection of plants from soil-borne diseases iseagerly anticipated, and biological control and other

non-chemical methods have received increasing atten-tion, for example, in control of tomato bacterial wilt.3–6)

From the viewpoint of protection of the naturalecological environment without the use of toxic chem-icals, slow sand filtration for exclusion of the influenceof pathogenic microorganisms is an important trial.3,5) Inthis study, we attempted to develop a new greenchemical method for controlling soil-borne plant dis-eases taking protection of the natural environmentcarefully into consideration, and investigated controlof tomato bacterial wilt caused by Ralstonia solanacea-rum using a novel methodology.

Our fundamental strategy includes three importantgreen principles. In the first place, we avoided dis-infection, because use of hazardous chemical fungicidesis not desirable for the protection of ecological environ-ment. In the second place, we avoided poorly biode-gradable chemical materials, because they show strongpersistence in soil and remain unchanged in the naturalenvironment for a long period. In the third place, wetried to reduce infectious contact between the roots ofplants and microbial cells in soil based on a novel andunique principle established by our research group. Forthis purpose, we used an equimolar copolymer of N-benzyl-4-vinylpyridinium chloride with styrene (PBVP-co-ST) that captures microbial cells alive on the surface.

We have reported capture of microbial cells in wateron the surface of cross-linked poly(N-benzyl-4-vinyl-pyridinium chloride) (PBVP) in a living state.7) Removalof bacteriophage8) and pathogenic human viruses9) bycapture on cross-linked PBVP, as well as removal ofmicroorganisms from water by filtration using non-woven cloth coated with PBVP-co-ST,10) have also beenreported. An electrochemical sensor for rapid determi-nation of viable aerobic microbial cell concentration has

y To whom correspondence should be addressed. Tel: +81-74-928-8363; Fax: +81-74-928-8528; E-mail: [email protected]

Abbreviations: PBVP, poly(N-benzyl-4-vinylpyridinium chloride); PBVP-co-ST, an equimolar copolymer of N-benzyl-4-vinylpyridinium chloride

with styrene; TTC, triphenyl tetrazolium chloride

Biosci. Biotechnol. Biochem., 69 (2), 326–333, 2005

been realized based on the consumption of oxygen byliving microbial cells captured on the surface of cross-linked PBVP.11) We performed a real-time record ofoxygen consumption using an oxygen electrode. Thisunique property of cross-linked PBVP and PBVP-co-STwas used to remove airborne microorganisms12) andviruses.13) Based on these experiences, we attempted tocapture pathogenic microorganisms in soil on thesurface of sawdust coated with PBVP-co-ST aiming toreduce infectious contact of the roots of plants withmicrobial cells. We did not anticipate disinfection bythis method because PBVP-co-ST captured microbialcells alive. In addition, we expected high biodegrad-ability for PBVP-co-ST, because cross-linked PBVPwas rapidly digested by activated sludge when placed inan aerobic treatment system for artificial sewage.14)

Materials and Methods

PBVP-co-ST. Commercial products of 4-vinylpyri-dine and styrene were purified as reported previously10)

before polymerization. Benzyl chloride, 2,20-azobisiso-butyronitrile, and other chemicals and solvents wereused without further purification. PBVP-co-ST wasprepared as described in a previous report.10) Intrinsicviscosity was 0.26 dl/g when determined in ethanol thatcontained 10 g/l of MgCl2.6H2O at 35 �C. In addition,copolymer of N-benzyl-4-vinylpyridinium chloride withstyrene in a molar ratio of 1:2 (PBVP-co-STa) was alsoprepared as a varied form of PBVP-co-ST with reducedbiodegradability. The weight average molecular weightdetermined by GPC measurement was 116,000. Sawdustcoated with 1wt% of PBVP-co-ST or PBVP-co-STawas used to control tomato bacterial wilt.

Ralstonia solanacearum. Commercial products oftriphenyl tetrazolium chloride (TTC), polymyxin Bsulfate, chloromycetin, polypeptone, casamino acid,crystal violet, and other chemicals were used withoutfurther purification. R. solanacearum E. F. Smith, strainOE1-1, was obtained from Dr. Ryo Ishikawa of TakedaChemical Industries, Ltd., Osaka, Japan. Cells wereprecultured at 30 �C for 3 d on agar plates that containedTTC medium prepared by dissolving polypeptone (10 g),casamino acid (1.0 g), sucrose (6.0 g), TTC (50mg),crystal violet (5mg), and agar (16 g) in 1,000mlsterilized distilled water. Pathogenic germ was distin-guished by the formation of white colonies and culturedin a growth medium prepared by dissolving polypeptone(10 g), casamino acid (1.0 g), and sucrose (6.0 g) in1,000ml sterilized distilled water where pH was adjustedto 7.0 before sterilization by autoclaving at 121 �C for20min. Cells were grown at 30 �C for 5 d with shaking,harvested by centrifugation at 13,000g for 20min, andwashed three times with sterilized distilled water.

Tomato. Seeds of tomato (Lycopersicon esculentumMill. cv. Momotaro) were purchased from Takii Seed

Corporation, Kyoto, Japan. They were used withoutfurther pretreatment. They were sowed on wet sterilizedabsorbent cotton that was placed in a laboratory dish.The dish was allowed to stand in a growth chamber forabout 7 d. After this procedure, young seedlings withtwo leaves were obtained and submitted for pot tests.The operation conditions of the growth chamber aredescribed below.

Pot tests in a growth chamber. Test soil for thedisease control was obtained from a farm of KyotoInstitute of Technology and mixed with 30wt% ofvermiculite. The mixture was sterilized by autoclavingat 121 �C for 180min. After 2 d, the autoclaving wasrepeated. Appropriate amounts of harvested cells ofR. solanacearum were suspended in sterilized distilledwater and added to a mixture of sterilized soil andcoated sawdust, and the mixture was placed in flower-pots. Each pot contained 500 g of the soil mixture.Young seedlings were transplanted into the soil andallowed to stand in a growth chamber. The chamber waskept at 27 �C with a 12-h photoperiod using fluorescentlamps. During the remaining 12 h, the growth chamberwas kept at 17 �C in the dark. Humidity was notcontrolled, and fluctuated between 75 and 85%. Thecontent of water in the test soil was kept to around120 g/kg. Diluted Hyponex solution was sprayed once aweek.The population of R. solanacearum in the soil was

measured as follows.15) Test soil (1.0 g) was sampledand added to sterilized distilled water (100ml), and themixture was stirred using an automatic mixer about10min, and was allowed to stand at room temperature.The bacterial cells crowded on to the surface of thecoated sawdust by the coagulation-like interactionbetween coated sawdust and bacterial cells, discussedbelow, and were dispersed during the mixing procedure.On the other hand, during the standing procedure, soilparticles and coated sawdust precipitated rapidly, andthe surface layer became transparent soon after. Afterseveral minutes of standing, the supernatant layer (thetransparent surface layer) was used for the measurementof viable cell count. The transparent supernatant layerdid not contain cells of R. solanacearum captured on thesurface of the coated sawdust, because the coatedsawdust was removed from the supernatant layer byprecipitation. Under these conditions, the amount ofcoated sawdust was 50 to 500mg/l, because the test soilcontained 5 to 50 g/kg of the coated sawdust. Thecoagulation-like interaction between coated sawdust andbacterial cells, discussed below, was not significantunder the diluted conditions. During cultivation, theamount of coated sawdust per liter of water was 42 to420 g, because the test soil contained 120 g/kg of water.Therefore, perhaps the population included bacterialcells crowded on to the surface of the coated sawdustdue to the coagulation-like interaction between thecoated sawdust and bacterial cells, but it did not contain

Control of Tomato Bacterial Wilt without Disinfection 327

the bacterial cells captured on the surface of the coatedsawdust. A 0.1-ml portion of the supernatant layer wasremoved and quickly mixed with 0.9ml of distilledwater, and then decimal serial dilutions were preparedfrom this by taking 0.1ml into 0.9ml of sterilizeddistilled water and mixing. From these dilutions, thesurviving R. solanacearum was counted on the TTCmedium by the spread-plate method. After inoculation,the plates were incubated at 30 �C, and the colonies werecounted after 3 d. The counting was done in quintupli-cate every time.After cultivation, the coated sawdust was isolated

from the test soil and washed with sterilized distilledwater. Electron micrographs of the cells of R. solana-cearum captured on the surface of isolated sawdust wererecorded using a Hitachi S-3200N scanning electronmicroscope.

Degradation of PBVP-co-ST by treatment with acti-vated sludge in soil. Biological degradation of PBVP-co-ST was performed by a treatment with activatedsludge in soil. Test soil was obtained from the shore ofLake Biwa, and was purified by washing with waterusing the Soxhlet extraction apparatus for 30 h. Thepurified soil was dried to constant weight before use. Apolymer sample (50mg) was dissolved in ethanol(10ml) and mixed with purified soil (50 g). The mixturewas then placed in a desiccator, and solvent wasremoved by drying under reduced pressure to constantweight.Activated sludge was obtained from domestic sewage

works immediately before biological degradation, andwas washed three times with sterilized physiologicalsaline. The purified activated sludge (0.71 g in wetweight corresponding to 50mg in dry weight) andartificial sewage16) with a COD concentration of1,000mg/l (10ml) were mixed with test soil. The totalamount of water in the test soil was kept around 150 g/kg. The mixture was allowed to stand at room temper-ature. After a prescribed time, the remaining polymerwas recovered by extraction with ethanol using theSoxhlet extraction apparatus for 100 h. Fine soil par-ticles contained in the extractive were removed bycentrifugation. The supernatant was placed in a rotaryevaporator, and solvents were removed by evaporation.Ethyl acetate was added to the residue and recoveredpolymer was precipitated. Low molecular weight or-ganic materials were removed by ethyl acetate. Theprecipitated polymer was isolated and dried to constantweight under reduced pressure. Prior to these experi-ments in biodegradation, a series of experiments wasperformed to ascertain the reliability of the recoveryprocedure.

Coagulation-like interaction between R. solanacea-rum and sawdust coated with PBVP-co-ST. Experimentswere performed under aseptic conditions at roomtemperature using a suspension (50ml) of R. solana-

cearum in sterilized physiological saline in a glass tube28mm in diameter and 20 cm long. Sawdust coated with1wt% of PBVP-co-ST or PBVP-co-STa (4.2 g) wasadded to the suspension, and the mixture was stirredusing an automatic mixer for 5min, and then allowed tostand at room temperature. After a prescribed time, thepopulation of R. solanacearum in the supernatant layerwas measured.

Results and Discussion

Control of tomato bacterial wilt using sawdust coatedwith PBVP-co-ST

Experiments in disease control were performed usingyoung seedlings of tomato and test soil that containedharvested cells of R. solanacearum and sawdust coatedwith 1wt% of PBVP-co-ST. The seedlings were trans-planted to test soil in flowerpots, and the pots wereplaced in a growth chamber.

The extent of disease was evaluated based onsymptoms of individual seedlings, and classified intofive categories: degree 0, no perceivable symptom wasrecognized; degree 1, symptom was limited only to tipof the seedling; degree 2, the whole body drooped butthe seedling stood straight; degree 3, the seedlingcollapsed but the body was still greenish and watery;degree 4, the seedling withered and whole body was dry.The degree of the symptom was closely related to thepopulation of R. solanacearum detected in roots. In thecase of degree 1, R. solanacearum was not alwaysdetected and population was less than 107 cfu/g. In thecase of degree 2, the population of R. solanacearum wasgenerally less than 108 cfu/g. In the case of degree 3, thepopulation was around 109 cfu/g. In the case of degree4, the population was generally larger than 109 cfu/g.

The extent of disease was evaluated based on thepercentage of seedlings that showed any degree ofsymptom, and the effect of disease control was eval-uated based on reduction in the percentage of symptoms.

In addition, the index of symptoms was defined asfollows, taking degree of symptom of an individualseedling into consideration:

Index of symptoms ¼ ½ðBþ 2Cþ 3Dþ 4EÞ

=4ðAþ Bþ Cþ Dþ EÞ� � 100

Here, A, B, C, D, and E are the numbers of seedlingsthat showed degrees of symptoms of 0, 1, 2, 3, and 4,respectively. In the case where all test seedlingscollapse, the index is 100. On the contrary, when nosymptom of disease is observed for any test seedlings,the index is 0. The effect of disease control wasevaluated based on reduction of the index of symptoms.

The addition of an appropriate amount of coatedsawdust controlled the disease. Figures 1 and 2 show thetime course of the percentage of symptoms and theindex of symptoms, respectively, during cultivation.Cross marks show the results obtained in the absence of

328 N. KAWABATA et al.

R. solanacearum and the coated sawdust. No symptomwas observed in this case, indicating that the cultivationconditions were suitable. On the other hand, closedcircles show the results obtained in the presence ofR. solanacearum but the absence of coated sawdust. Thepercentage of symptoms and the index of symptomsincreased with time and reached 75% and 75 respec-tively after 6 weeks. These results indicate that the testsoil was suitable as a model soil contaminated byR. solanacearum.

Closed triangles show the results obtained in thepresence of R. solanacearum and 10 g/kg of non-coatedsawdust. Obviously, non-coated sawdust did not exhibitcontrol of the disease at all. On the other hand, opentriangles indicate that addition of 5 g/kg of the coatedsawdust effected a 40% reduction in appearance and a24% reduction in the index of symptoms, respectively,after 6 weeks. Open circles indicate that the addition of10 g/kg of coated sawdust effected a 92% reduction inappearance and a 87% reduction in the index ofsymptoms, respectively, after 6 weeks. Open squaresindicate that the addition of 50 g/kg of coated sawdusteffected a 49% reduction in appearance and a 31%reduction in the index of symptoms, respectively, after 6weeks. The addition of 10 g/kg of coated sawdust wasmost appropriate. In this case, the amount of PBVP-co-ST added to the test soil was 100mg/kg.

Figures 1 and 2 indicate that the addition of coatedsawdust predominantly effected reduction in the per-centage of symptoms. This result suggests that thecoated sawdust effected control of the soil-borne disease

mainly due to the reduction of infectious contactbetween the roots of tomato and cells of R. solanacea-rum rather than restraining of progress of the symptomof individual seedlings.Figure 3 shows an electron micrograph of the coated

sawdust isolated from test soil after cultivation where10 g/kg of the coated sawdust was used. Sawdust coatedwith PBVP-co-ST captured cells of R. solanacearum onthe surface, similarly to the case of non-woven clothcoated with PBVP-co-ST.10)

Population of R. solanacearum in soil during culti-vationFigure 4 shows the time course of the population of

R. solanacearum in soil that was not captured on thesurface of the coated sawdust. Closed circles show theexperimental results obtained in the absence of thecoated sawdust. Open triangles indicate that the addition

Fig. 1. Time Course of the Percentage of Symptoms in the Control of

Tomato Bacterial Wilt Caused by R. solanacearum Using Sawdust

Coated with 1wt% of PBVP-co-ST.

The number of test seedlings for each experiment is given in

parentheses: (�), performed in the absence of R. solanacearum and

coated sawdust (120); ( ), performed in the presence of R. sola-

nacearum but the absence of coated sawdust (91); ( ), performed in

the presence of R. solanacearum and 10 g/kg of non-coated sawdust

(40); ( ), performed in the presence of R. solanacearum and 5 g/kg

of coated sawdust (102); ( ), performed in the presence of

R. solanacearum and 10 g/kg of coated sawdust (92); ( ), per-

formed in the presence of R. solanacearum and 50 g/kg of coated

sawdust (42).

Fig. 2. Time Course of Index of Symptoms in the Control of Tomato

Bacterial Wilt Caused by R. solanacearum Using Sawdust Coated

with 1wt% of PBVP-co-ST.

Same as in Fig. 1.

Fig. 3. Electron Micrograph of Coated Sawdust Isolated from Test

Soil after Cultivation.

In this case, 10 g/kg of sawdust coated with 1wt% of PBVP-co-

ST was used.


of 5 g/kg of coated sawdust reduced the population toabout 1/3. At a glance, the disease control can beexplained in terms of the reduction of populationeffected by the addition of coated sawdust.However, the open circles of Fig. 4 indicate that the

addition of 10 g/kg of the coated sawdust did not exhibita significant influence on the population, althoughtomato bacterial wilt was dramatically controlled underthese conditions. Furthermore, the open squares ofFig. 4 indicate that the addition of 50 g/kg of the coatedsawdust resulted in about a 50% increase in thepopulation, in spite of the fact that tomato bacterialwilt was controlled to some extent under these con-ditions.Thus, further investigation was required to understand

the reason for the disease control effected by theaddition of the coated sawdust. The population ofR. solanacearum that was not captured by the coatedsawdust increased with the amount of coated sawdustadded to the test soil. The population was in the order:open squares (obtained in the presence of 50 g/kg ofcoated sawdust) > open circles (obtained in the pres-ence of 10 g/kg of coated sawdust) > open triangles(obtained in the presence of 5 g/kg of coated sawdust).This phenomenon can be explained in terms of the highbiodegradability of PBVP-co-ST, i.e., R. solanacearumappeared to consume PBVP-co-ST as a component ofnutrient due to high biodegradability, and resulted inproliferation.When 5 g/kg of coated sawdust was added, capture of

the microbial cells by the coated sawdust predominatedover proliferation of the bacteria effected by PBVP-co-ST. However, when 50 g/kg of the coated sawdust wasadded, the proliferation effected by PBVP-co-ST greatlypredominated over capture of bacterial cells by thecoated sawdust. In the case where 10 g/kg of the coated

sawdust was added, the two phenomena appeared to bebalanced.

Biodegradation of PBVP-co-ST by treatment withactivated sludge in soil

Biodegradation of PBVP-co-ST was performed bytreatment with activated sludge in soil at room temper-ature. Figure 5 shows the time course of weightreduction during treatment. The residual weight is thetotal amount of polymeric materials recovered aftertreatment. The weight reduction followed first-orderkinetics. The half-life of the degradation was 5.6 d, andsuggested that 99.9% of PBVP-co-ST disappears during2 months of the biological treatment. Although biode-gradation of PBVP-co-ST in the natural environmentmight require a much more prolonged period, it is notnecessary to fear severe persistence for PBVP-co-ST inthe natural environment. Further research is required toelucidate the influence of degradation products derivedfrom PBVP-co-ST on the ecological system beforepractical application of the new methodology. However,since microorganisms involved in activated sludge arefond of PBVP-co-ST and digest it rapidly, and it is notnecessary fear severe persistence in the environment,PBVP-co-ST is hopefully harmonious with the naturalenvironment.

Control of tomato bacterial wilt using sawdust coatedwith a varied form of PBVP-co-ST with reducedbiodegradability

Too much biodegradability of PBVP-co-ST is notdesirable in the control of soil-borne plant disease,because it accompanies proliferation of microorganismsin the soil. Hence, we attempted to use PBVP-co-STa, acopolymer of N-benzyl-4-vinylpyridinium chloride withstyrene in a molar ratio of 1:2, as a varied form ofPBVP-co-ST with reduced biodegradability.

Fig. 4. Time Course of the Population of R. solanacearum in Soil

That Was Not Captured by Sawdust Coated with 1wt% of PBVP-

co-ST.

( ), performed in the absence of coated sawdust; ( ), performed

in the presence of 5 g/kg of coated sawdust; ( ), performed in the

presence of 10 g/kg of coated sawdust; ( ), performed in the

presence of 50 g/kg of coated sawdust.

0

20

40

60

80

100

0 10 20 30

Time (d)

Res

idua

l wei

ght (

%)

Fig. 5. Time Course of Weight Reduction in PBVP-co-ST during

Treatment with Activated Sludge in Soil at Room Temperature.

Initial amount of PBVP-co-ST, 1.0 g/kg.; amount of artificial

sewage added to assist biodegradation, 0.2 g/kg in COD; activated

sludge, 14.2 g/kg in wet weight; water, 150 g/kg.


Biodegradation of PBVP-co-STa was performedunder the same conditions as for PBVP-co-ST. Theresidual weight after 2 weeks of treatment was 42%, andthe half-life was about 10 d. Enrichment of styrene inPBVP-co-ST to prepare PBVP-co-STa certainly reducedbiodegradability.

Control of tomato bacterial wilt by the addition ofsawdust coated with 1wt% of PBVP-co-STa wasperformed in a similar manner. When 10 g/kg of thecoated sawdust was added, the population of R. solana-cearum in soil that was not captured by the coatedsawdust was less than 107 cfu/g, much less than that inthe corresponding experiments for PBVP-co-ST, indi-cated by open circles in Fig. 4, probably due to thereduced biodegradability of PBVP-co-STa. Contrary toour expectations, however, control of the disease bysawdust coated with PBVP-co-STa was not satisfactory.For example, the addition of 10 g/kg of the coatedsawdust effected a 25% reduction in appearance and an11% reduction in the index of symptoms, respectively,after 6 weeks. The addition of 20 g/kg of coated sawdustimproved on this to some extent, but resulted in a 49%reduction in appearance and a 65% reduction in theindex of symptoms, respectively, after 6 weeks, al-though proliferation of R. solanacearum was not verysignificant in this case. Enrichment of styrene in PBVP-co-ST probably reduced the ability to capture microbialcells on the surface and resulted in reduced preventionof infectious contact between the roots of plants andmicrobial cells in soil, and gave unsatisfactory control ofthe disease.

Coagulation-like interaction between R. solanacea-rum and sawdust coated with PBVP-co-ST

It is difficult to explain the disease control by thecoated sawdust in terms of reduction of the populationof R. solanacearum in soil that was not captured by thecoated sawdust. As Figs. 1 and 2 indicate, the additionof 10 g/kg of the coated sawdust was most appropriatefor disease control. However, as Fig. 4 indicates, thepopulation of non-captured R. solanacearum underthese conditions (open circles) was close to that in theabsence of the coated sawdust (closed circles). AsFigs. 1 and 2 indicate, the addition of 50 g/kg of coatedsawdust effected a 49% reduction in appearance and a31% reduction in the index of symptoms after 6 weeks,but as Fig. 4 indicates, the population of non-capturedR. solanacearum (open squares) was much higher thanthat in the absence of the coated sawdust (closedcircles). Therefore, we suspected another unknowninteraction between cells of R. solanacearum and thecoated sawdust, and investigated the interaction in theabsence of soil. Since we used 10 g/kg of the coatedsawdust under the most appropriate conditions, and thetest soil contained about 120 g/kg of water, weperformed a series of additional experiments usingharvested cells of R. solanacearum suspended in 50mlof sterilized physiological saline and 4.2 g of sawdust

coated with 1wt% of PBVP-co-ST or PBVP-co-STa.In a test tube, coated sawdust was added to a

suspension of R. solanacearum in sterilized physiolog-ical saline. The mixture was vigorously stirred for 5min,and was kept standing at room temperature. After aprescribed time, the population of R. solanacearum inthe supernatant layer was measured. The time course ofthe supernatant population is shown in Fig. 6. Closedcircles indicate the data obtained in the absence ofcoated sawdust. No significant change in population wasobserved. On the contrary, open circles indicate the dataobtained in the presence of sawdust coated with PBVP-co-STa. The addition of coated sawdust effected aremarkable reduction in the supernatant population. Therate of reduction of the population was about 1/100. Onthe other hand, in the case where sawdust coated withPBVP-co-ST was added, R. solanacearum was notdetected in the supernatant layer. In this case, thesupernatant population was less than 102 cfu/ml. Therate of reduction in the population was assumed to be 1/1000 or less.We found sedimentation of visually recognizable thin

flocks of R. solanacearum on the surface of the coatedsawdust. This phenomenon has been overlooked inprevious studies on capture of microbial cells on thesurface of beads of cross-linked PBVP7,11) or celluloseor non-woven cloth coated with PBVP-co-ST.10,12)

Visual recognition of microbial cells captured on thesesurfaces is impossible, because the microbial cells werecaptured in a monolayer state. Therefore, visuallyrecognizable flocks appeared to be crowded cells ofR. solanacearum. Formation of visible flocks settled onthe surface of sawdust coated with PBVP-co-ST closelyresembled coagulation of microbial cells by PBVP.17) Inthis report, therefore, sedimentation of bacterial flockson the surface of coated sawdust is called ‘‘coagulation-

2

3

4

5

6

0 5 10

Time (h)

Loga

rithm

of p

opul

atio

n (c

fu/m

l)

Fig. 6. Time Course of the Supernatant Population of R. solanacea-

rum Suspended in Sterilized Physiological Saline (50ml) in the

Absence ( ) and the Presence ( ) of 4.2 g of Sawdust Coated with

1wt% of PBVP-co-STa.

In the presence of 4.2 g of sawdust coated with 1wt% of PBVP-

co-ST, R. solanacearum was not detected in the supernatant layer,

and the supernatant population appeared to be less than 102 cfu/ml.


like interaction’’ between bacterial cells and coatedsawdust.The state of microbial cells in flocks formed by

coagulation using PBVP17) and the ‘‘coagulation-likeinteraction’’ is completely different from that of micro-bial cells captured on the surface of cross-linkedPBVP.7) Flocks formed by coagulation17) or the ‘‘coag-ulation-like interaction’’ were easily loosened by shak-ing the glass tube. On the contrary, it is extremelydifficult to remove captured microbial cells from thesurface of cross-linked PBVP.7) The capacity of cross-linked PBVP to capture microbial cells on the surfaceshowed limitations due to surface area, and everypossible effort to restore the ability to capture microbialcells again ended in failure. Therefore, it is quitereasonable to consider that the population shown inFig. 4 includes bacterial cells settled on the surface ofthe coated sawdust due to the ‘‘coagulation-like inter-action’’, but does not include bacterial cells captured onthe surface of the coated sawdust.As Fig. 4 indicates, the population observed in the

presence of 10 g/kg of coated sawdust (open circles)was close to that observed in the absence of coatedsawdust (closed circles), although tomato bacterial wiltwas dramatically controlled by the addition of 10 g/kgof coated sawdust (Figs. 1 and 2, open circles and closedcircles). Studies on the interaction between the coatedsawdust and the bacterial cells suggested that thepopulation of free cells in soil was reduced at least toless than 1/1000 when 10 g/kg of the coated sawdustwas added. Thus, the disease control can be explained interms of the coagulation-like interaction.As Fig. 4 indicates, the population observed in the

presence of 50 g/kg of the coated sawdust (opensquares) was 30 to 70% larger than that observed inthe absence of the coated sawdust (closed circles),although tomato bacterial wilt was controlled to someextent by the addition of 50 g/kg of coated sawdust(Figs. 1 and 2, open squares and closed circles). Studieson the interaction between the coated sawdust and thebacterial cells suggested that the population of free cellsin soil was reduced at least to less than 1/1000 when50 g/kg of the coated sawdust was added.These observations prompted us to consider that the

‘‘coagulation-like interaction’’ between the coated saw-dust and cells of R. solanacearum plays an importantrole in the control of tomato bacterial wilt, in addition tothe capture of bacterial cells on the surface of the coatedsawdust, as shown in Fig. 3. This coagulation-likeinteraction might reduce infectious contact of roots ofplants with cells of R. solanacearum in soil. Sawdustcoated with PBVP-co-ST indicated stronger coagula-tion-like interaction against bacterial cells than sawdustcoated with PBVP-co-STa, in accordance with the resultthat the former coated sawdust indicated more effectivecontrol of soil-borne disease.The excellent biodegradability of PBVP-co-ST is

desirable for use in the natural environment, because it is

not necessary to be afraid of severe persistence in soil.However, the biodegradability accompanies prolifera-tion of microorganisms in soil, and we must avoid theaddition of too much of the coated sawdust. The mostappropriate amount of coated sawdust should bedetermined taking the balance of the two opposedeffects carefully into consideration. The next step in thisresearch is an examination of the practical utility of thenew green chemical methodology in the field.

Acknowledgments

The authors are grateful to Dr. Ryo Ishikawa for hiskind contribution of the strain of R. solanacearum weused, and to Professor Emeritus Iwao Furusawa ofKyoto University for helpful advice and discussion.

References

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control of tomato bacterial wilt without disinfection using a new functional polymer that captures...

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