Inoculum effect on the efficacies of amoxicillin-clavulanate, piperacillin-tazobactam, and imipenem against extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing Escherichia coli in an experimental murine sepsis model.

Escherichia coli is commonly involved in infections with a heavy bacterial burden. Piperacillin-tazobactam and carbapenems are among the recommended empirical treatments for health care-associated complicated intra-abdominal infections. In contrast to amoxicillin-clavulanate, both have reduced in vitro activity in the presence of high concentrations of extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing E. coli bacteria. Our goal was to compare the efficacy of these antimicrobials against different concentrations of two clinical E. coli strains, one an ESBL-producer and the other a non-ESBL-producer, in a murine sepsis model. An experimental sepsis model {~5.5 log10 CFU/g [low inoculum concentration (LI)] or ~7.5 log(10) CFU/g [high inoculum concentration (HI)]} using E. coli strains ATCC 25922 (non-ESBL producer) and Ec1062 (CTX-M-14 producer), which are susceptible to the three antimicrobials, was used. Amoxicillin-clavulanate (50/12.5 mg/kg given intramuscularly [i.m.]), piperacillin-tazobactam (25/3.125 mg/kg given intraperitoneally [i.p.]), and imipenem (30 mg/kg i.m.) were used. Piperacillin-tazobactam and imipenem reduced spleen ATCC 25922 strain concentrations (-2.53 and -2.14 log10 CFU/g [P < 0.05, respectively]) in the HI versus LI groups, while amoxicillin-clavulanate maintained its efficacy (-1.01 log10 CFU/g [no statistically significant difference]). Regarding the Ec1062 strain, the antimicrobials showed lower efficacy in the HI than in the LI groups: -0.73, -1.89, and -1.62 log10 CFU/g (P < 0.05, for piperacillin-tazobactam, imipenem, and amoxicillin-clavulanate, respectively, although imipenem and amoxicillin-clavulanate were more efficacious than piperacillin-tazobactam). An adapted imipenem treatment (based on the time for which the serum drug concentration remained above the MIC obtained with a HI of the ATCC 25922 strain) improved its efficacy to -1.67 log10 CFU/g (P < 0.05). These results suggest that amoxicillin-clavulanate could be an alternative to imipenem treatment of infections caused by ESBL- and non-ESBL-producing E. coli strains in patients with therapeutic failure with piperacillin-tazobactam.

Determination of an inoculum effect with various cephalosporins among clinical isolates of methicillin-susceptible Staphylococcus aureus.

Using 98 clinical methicillin-susceptible Staphylococcus aureus isolates of known beta-lactamase (Bla) type, we found a pronounced inoculum effect for cephalexin (mostly Bla type A and C strains), a mild inoculum effect for cephalothin (especially types B and C), and no inoculum effects for ceftriaxone and cefuroxime. Ceftobiprole showed the lowest MICs at a high inoculum but with a slight increase for Bla-positive versus Bla-negative strains. Since a potential therapeutic effect associated with a cephalosporin inoculum effect has been described, further studies are warranted.

Inoculum effect with cefazolin among clinical isolates of methicillin-susceptible Staphylococcus aureus: frequency and possible cause of cefazolin treatment failure.

Methicillin (meticillin)-susceptible Staphylococcus aureus (MSSA) strains producing large amounts of type A beta-lactamase (Bla) have been associated with cefazolin failures, but the frequency and impact of these strains have not been well studied. Here we examined 98 MSSA clinical isolates and found that 26% produced type A Bla, 15% type B, 46% type C, and none type D and that 13% lacked blaZ. The cefazolin MIC(90) was 2 microg/ml for a standard inoculum and 32 microg/ml for a high inoculum, with 19% of isolates displaying a pronounced inoculum effect (MICs of >or=16 microg/ml with 10(7) CFU/ml) (9 type A and 10 type C Bla producers). At the high inoculum, type A producers displayed higher cefazolin MICs than type B or C producers, while type B and C producers displayed higher cefamandole MICs. Among isolates from hemodialysis patients with MSSA bacteremia, three from the six patients who experienced cefazolin failure showed a cefazolin inoculum effect, while none from the six patients successfully treated with cefazolin showed an inoculum effect, suggesting an association between these strains and cefazolin failure (P = 0.09 by Fisher's exact test). In summary, 19% of MSSA clinical isolates showed a pronounced inoculum effect with cefazolin, a phenomenon that could explain the cases of cefazolin failure previously reported for hemodialysis patients with MSSA bacteremia. These results suggest that for serious MSSA infections, the presence of a significant inoculum effect with cefazolin could be associated with clinical failure in patients treated with this cephalosporin, particularly when it is used at low doses.

The Effect of Inoculum Size on Selection of In Vitro Resistance to Vancomycin, Daptomycin, and Linezolid in Methicillin-Resistant Staphylococcus aureus.

OBJECTIVES: The inoculum effect (IE) is an increase in the minimum inhibitory concentration (MIC) at high bacterial densities. The effect of three inoculum sizes on the selection of resistance to vancomycin, daptomycin, and linezolid was investigated in methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Low (10(4) CFU/ml), medium (10(6) CFU/ml), and high (10(8) CFU/ml) inocula of MRSA were exposed to twofold increasing concentrations of either drug during 15 days of cycling. MICs for low (MICL), medium (MICM), and high (MICH) inocula were determined daily. Conventional MICs were measured at days 1, 5, 10, and 15. Experiments were performed in triplicate. RESULTS: At the beginning of the experiment a small IE was observed for vancomycin (MICL=1 μg/ml, MICM=1-2 μg/ml, and MICH=2 μg/ml) and a significant IE for daptomycin (MICL=0.25 μg/ml, MICM=0.25-0.5 μg/ml, and MICH=2 μg/ml). Linezolid exhibited no IE at low and medium inocula (MICL=1 μg/ml and MICM=1-2 μg/ml), but with the high inoculum, concentrations up to 2,048 μg/ml did not fully inhibit visual growth. During cycling, increase of MIC was observed for all antibiotics. At day 15, MICL, MICM, and MICH of vancomycin were 2-4, 4-8, and 4-16 μg/ml and of daptomycin were 0.5-2, 8-128, and 64-256 μg/ml, respectively. MICL and MICM of linezolid were 1 and 2-4 μg/ml, respectively. Conventional MICs showed vancomycin and daptomycin selection of resistance since day 5 depending on the inocula. No selection of linezolid resistance was observed. CONCLUSIONS: Our results showed the importance of the inoculum size in the development of resistance. Measures aimed at lowering the inoculum at the site of infection should be used whenever possible in parallel to antimicrobial therapy.

New methods for detecting antibiotic resistant bacteria and preventing their spread

Methicillin resistant Staphylococcus aureus (MRSA) bacteria have emerged in the early 1980's in numerous health care institutions around the world. The main transmission mechanism within hospitals and healthcare facilities is through the hands of health care workers. Resistant to several antibiotics, the MRSA is one of the most feared pathogens in the hospital setting since it is very difficult to eradicate with the standard treatments. There are still a limited number of anti-MRSA antibiotics but the first cases of resistance to these compounds have already been reported and their frequency is likely to increase in the coming years. Every year, the MRSA infections result in major human and financial costs, due to the high associated mortality and expenses related to the required care. Measures towards a faster detection of resistant bacteria and establishment of appropriate antibiotic treatment parameters are fundamental. Also as part as infection prevention, diminution of bacteria present on the commonly touched surfaces could also limit the spread and selection of antibiotic resistant bacteria. During my thesis, projects were developed around MRSA and antibiotic resistance investigation using innovative technologies. The thesis was subdivided in three main parts with the use of atomic force microscopy AFM for antibiotic resistance detection in part 1, the importance of the bacterial inoculum size in the selection of antibiotic resistance in part 2 and the testing of antimicrobial surfaces creating by sputtering copper onto polyester in part 3. In part 1 the AFM was used two different ways, first for the measurement of stiffness (elasticity) of bacteria and second as a nanosensor for antibiotic susceptibility testing. The stiffness of MRSA with different susceptibility profiles to vancomycin was investigated using the stiffness tomography mode of the AFM and results have demonstrated and increased stiffness in the vancomycin resistant strains that also paralleled with increased thickness of the bacterial cell wall. Parts of the AFM were also used to build a new antibiotic susceptibility-testing device. This nano sensor was able to measure vibrations emitted from living bacteria that ceased definitively upon antibiotic exposure to which they were susceptible but restarted after antibiotic removal to which they were resistant, allowing in a matter of minute the assessment of antibiotic susceptibility determination. In part 2 the inoculum effect (IE) of vancomycin, daptomycin and linezolid and its importance in antibiotic resistance selection was investigated with MRSA during a 15 days of cycling experiment. Results indicated that a high bacterial inoculum and a prolonged antibiotic exposure were two key factors in the in vitro antibiotic resistance selection in MRSA and should be taken into consideration when choosing the drug treatment. Finally in part 3 bactericidal textile surfaces were investigated against MRSA. Polyesters coated after 160 seconds of copper sputtering have demonstrated a high bactericidal activity reducing the bacterial load of at least 3 logio after one hour of contact. -- Au cours des dernières décennies, des bactéries multirésistantes aux antibiotiques (BMR) ont émergé dans les hôpitaux du monde entier. Depuis lors, le nombre de BMR et la prévalence des infections liées aux soins (IAS) continuent de croître et sont associés à une augmentation des taux de morbidité et de mortalité ainsi qu'à des coûts élevés. De plus, le nombre de résistance à différentes classes d'antibiotiques a également augmenté parmi les BMR, limitant ainsi les options thérapeutiques disponibles lorsqu'elles ont liées a des infections. Des mesures visant une détection plus rapide des bactéries résistantes ainsi que l'établissement des paramètres de traitement antibiotiques adéquats sont primordiales lors d'infections déjà présentes. Dans une optique de prévention, la diminution des bactéries présentes sur les surfaces communément touchées pourrait aussi freiner la dissémination et l'évolution des bactéries résistantes. Durant ma thèse, différents projets incluant des nouvelles technologies et évoluant autour de la résistance antibiotique ont été traités. Des nouvelles technologies telles que le microscope à force atomique (AFM) et la pulvérisation cathodique de cuivre (PCC) ont été utilisées, et le Staphylococcus aureus résistant à la méticilline (SARM) a été la principale BMR étudiée. Deux grandes lignes de recherche ont été développées; la première visant à détecter la résistance antibiotique plus rapidement avec l'AFM et la seconde visant à prévenir la dissémination des BMR avec des surfaces crées grâce à la PCC. L'AFM a tout d'abord été utilisé en tant que microscope à sonde locale afin d'investiguer la résistance à la vancomycine chez les SARMs. Les résultats ont démontré que la rigidité de la paroi augmentait avec la résistance à la vancomycine et que celle-ci corrélait aussi avec une augmentation de l'épaisseur des parois, vérifiée grâce à la microscopie électronique. Des parties d'un AFM ont été ensuite utilisées afin de créer un nouveau dispositif de test de sensibilité aux antibiotiques, un nanocapteur. Ce nanocapteur mesure des vibrations produites par les bactéries vivantes. Après l'ajout d'antibiotique, les vibrations cessent définitivement chez les bactéries sensibles à l'antibiotique. En revanche pour les bactéries résistantes, les vibrations reprennent après le retrait de l'antibiotique dans le milieu permettant ainsi, en l'espace de minutes de détecter la sensibilité de la bactérie à un antibiotique. La PCC a été utilisée afin de créer des surfaces bactéricides pour la prévention de la viabilité des BMR sur des surfaces inertes. Des polyesters finement recouverts de cuivre (Cu), connu pour ses propriétés bactéricides, ont été produits et testés contre des SARMs. Une méthode de détection de viabilité des bactéries sur ces surfaces a été mise au point, et les polyesters obtenus après 160 secondes de pulvérisation au Cu ont démontré une excellente activité bactéricide, diminuant la charge bactérienne d'au moins 3 logio après une heure de contact. En conclusion, l'utilisation de nouvelles technologies nous a permis d'évoluer vers de méthodes de détection de la résistance antibiotique plus rapides ainsi que vers le développement d'un nouveau type de surface bactéricide, dans le but d'améliorer le diagnostic et la gestion des BMR.

Resistance to nucleoside analog reverse transcriptase inhibitors mediated by human immunodeficiency virus type 1 p6 protein.

Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within the pol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6(gag)-p6(pol) region of HIV-1, placed immediately upstream of pol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6(Gag)), or by changes in activation of the viral protease (p6(Pol)). Duplication of the proline-rich p6(Gag) PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naïve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6(Pol)), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

Effect of the concentration of inoculum and tensoactive on the efficiency of Bipolaris sorokiniana infection in wheat leaves

Techniques that result in increased pathogen infection rates by employing reduced quantities of fungal spores with sparse sporulation have been developed. Experiments under controlled environment conditions were conducted to evaluate the effect of the density of Bipolaris sorokiniana conidia on the intensity of wheat helminthosporiosis. Using a selected inoculum density, the concentration of the tensoactive (Tween 20) that promoted maximum infection by the causal agent of the disease was determined. The density of lesions and the estimated severity of the disease were quantified. The selected inoculum density was 1.5 x 10(4) spores.mL-1 plus 480 µL tensoactive.L-1 water, resulting in a disease severity that allows selecting wheat cultivars resistant to B. sorokiniana.

Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials.

EFFECT OF CULTIVATION CONDITIONS ON GLUCOSE-6-PHOSPHATE DEHYDROGENASE PRODUCTION BY GENETICALLY MODIFIED Saccharomyces cerevisiae

The objective of this research was to improve Glucose-6-phosphate dehydrogenase (G6PD) production by Saccharomyces cerevisiae W303-181, which carry the plasmid YEpPGK-G6PD, by varying the following cultivation conditions: pH value (4.8, 5.7 and 6.6); inoculum concentration (0.1, 0.6 and 1.1 g/L) and initial glucose concentration (20.0, 30.0 and 40.0 g/L). The effect of those variables on G6PD production capability was studied by the application of response surface statistical analysis. The results showed that the highest G6PD production (1594.2 U/L), specific activity (1189.7 U/g(cell)) and productivity (45.6 U/L.h) occurred at pH 4.8, inoculum concentration of 0.1 g/L and initial glucose concentration of 20.0 g/L, under agitation of 150 rpm at 30 degrees C after 36 h. In this work, the strain expressed about 21 fold more activity than the wild S. cerevisiae strain, being an attractive and promising new source of this enzyme.

Protective effect of carbon dioxide against bacterial peritonitis induced in rats

Carbon dioxide (CO(2)) has been used in the food industry as an antimicrobial agent. This study aimed to investigate whether CO(2) pneumoperitoneum might act similarly as an antimicrobial agent in the infected peritoneal cavity. Peritonitis was induced in 58 rats by intraabdominal injection of an Escherichia coli inoculum (6 x 105 colony-forming units [CFU]/ml). Control rats were injected with saline solution. The rats were randomly divided into four groups: rat control (RC, n = 15), bacterial inoculation control (BIC, n = 10), bacterial inoculation and laparotomy (BIL, n = 17), and bacterial inoculation and CO(2) pneumoperitoneum (BIP, n = 16). The survival rates and histopathologic changes in the abdominal wall muscles, spleen, liver, intestines, and omentum were evaluated, and the samples were classified as ""preserved"" or ""inflamed"" (acute inflammation or tissue regeneration). The survival rates for the four groups were as follows: RC (100%), BIP (75%), BIL (53%), and BIC (30%). With regard to survival rates, statistically significant differences were observed between the following groups: RC and BIC (p = 0.0009), RC and BIL (p = 0.0045), BIP and BIC (p = 0.0332), and RC and BIP (p = 0.0470). No significant differences regarding survival rates were observed between the BIL and BIC groups or between the BIP and BIL groups. With regard to the number of inflamed samples per group, a statistically significant difference was observed between the BIC and RC groups and the BIL and RC groups (p = 0.05). Carbon dioxide pneumoperitoneum has a protective effect against bacterial peritonitis induced in rats.

The effect of mode of exposure to Beauveria bassiana on conidia acquisition and host mortality of Colorado potato beetle, Leptinotarsa decemlineata

The effects of the mode of exposure of second instar Colorado potato beetles to Beauveria bassiana on conidia acquisition and resulting mortality were investigated in laboratory studies. Larvae sprayed directly with a B, bassiana condial suspension, larvae exposed to B, bassiana-treated foliage, and larvae both sprayed and exposed to treated foliage experienced 76, 34, and 77% mortality, respectively. The total number of conidia and the proportion of germinating conidia were measured over time for four sections of the insect body: the ventral surface of the head (consisting mostly of ventral mouth parts), the ventral abdominal surface, the dorsal abdominal surface, and the legs. From observations at 24 and 36 h posttreatment, mean totals of 161.1 conidia per insect were found on sprayed larvae, 256.1 conidia on larvae exposed only to treated foliage, and 408.3 conidia on larvae both sprayed and exposed to treated foliage, On sprayed larvae, the majority of conidia were found on the dorsal abdominal surface, whereas conidia were predominantly found in the ventral abdominal surface and mouth parts on larvae exposed to treated foliage, Between 24 and 36 h postinoculation the percentage of conidia germinating on sprayed larvae increased slightly from 80 to 84%), On the treated foliage, the percentage of germinated conidia on larvae increased from 35% at 24 h to 50% at 36 h posttreatment, Conidia germination on sprayed larvae on treated foliage was 65% at 24 h and 75% at 36 h posttreatment, It is likely that the gradual acquisition of conidia derived from the continuous exposure to B. bassiana inoculum on the foliar surface was responsible for the increase in germination over time on larvae exposed to treated foliage, The density and germination of conidia were observed 0, 4, 8, 12, 16, 20, and 24 h after being sprayed with or dipped in conidia suspensions or exposing insects to contaminated foliage, Conidia germinated twice as fast on sprayed insects as with any other treatment within the first 12 h, This faster germination may be due to the pressure of the sprayer enhancing conidial lodging on cuticular surfaces. (C) 2001 Academic Press.

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of tine of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can he reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the V formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Effect of fertilizer-N application and seed coating with rhizobial inoculants on soybean yield in eastern Paraguay

Nitrogen removal in soybean grains at harvest may exceed biological N2 fixation, particularly if grain yields are as high as typically achieved on "Terra Rossa" soils of Eastern Paraguay. Applying N fertilizer or coating seeds with rhizobial inoculants that enhance nodulation may represent a way of balancing the N budget. However, the effects of such treatments appear to be highly site-specific. The objective of this study was to examine the effects of N application (N) and rhizobial inoculation (I) on nodulation, N accumulation and soybean yields in Eastern Paraguay. Field experiments were conducted in two consecutive soybean seasons. Dry conditions in the first year delayed sowing and reduced plant number m-2 and pod number plant-1. Grain yields were generally below 2 t ha-1 but the +N+I treatment increased yields by about 75%. In the second year favorable conditions resulted in yields of around 4 t ha-1 and the treatments had no effect. Nitrogen accumulation was higher in the first year and could therefore not explain the observed yield differences between years and treatment combinations. The positive effect of the +N+I treatment in year one was associated with a more rapid root growth which could have reduced susceptibility to intermittent drought stress. Nodule biomass decreased between flowering and pod setting stages in the +I treatment whereas further increases in nodule biomass in the -I treatment may have led to competition for assimilates between nodules and developing pods. Based on these preliminary results we conclude that N application and seed inoculation can offer short-term benefits in unfavorable years without negative effects on yield in favorable years.

Effect of organic amendments on sclerotial germination, mycelial growth, and Sclerotium rolfsii-induced diseases

The addition of organic residues to soil is an option to control some soil-borne diseases. Benzaldehyde and powders of kudzu (Pueraria lobata), velvetbean (Mucuna deeringiana), and pine-bark (Pinus elliottii and P. taeda) added to soil could reduce certain soil-borne diseases. This study evaluated the effects of benzaldehyde and the dried powders of kudzu, velvetbean, and pine-bark as soil amendments on germination and formation of sclerotia, on mycelial growth of Sclerotium rolfsii, on plant survival, and disease incidence. The data showed that high amounts of benzaldehyde (0.4 ml kg-1 of soil) and velvetbean (100 g kg-1) inhibited S. rolfsii mycelial growth and sclerotium germination. However, low amounts of benzaldehyde (0.1 ml kg-1), kudzu (25 g kg-1), and pine-bark (25 g kg-1) stimulated mycelial growth and sclerotium germination. Kudzu (25-100 g kg-1) and velvetbean (25-100 g kg-1) inhibited the formation of sclerotia. Nevertheless, benzaldehyde at 0.2 and 0.4 ml kg-1 stimulated the formation of sclerotia. Kudzu (50 and 100 g kg-1) and pine-bark (50 g kg-1) favored the colonization of sclerotia by Trichoderma sp. The numbers of soybean (Glycine max) plants were higher and diseased plants were lower than the non-amend soil in the following treatments: kudzu (50 and 100 g kg-1), velvetbean (50 and 100 g kg-1), and pine-bark (50 g kg-1). Disease severity on tomato (Lycopersicon esculentum) plants was low in soil treated with kudzu or velvetbean (30 and 35 g kg-1) and pine-bark (35 g kg-1). Dried powders of kudzu, velvetbean, or pine-bark added to soil can reduce disease by reducing pathogen inoculum.