Patent Application: US-91083404-A

Abstract:
strains of rhizobium leguminosarum biovar viceae have antifungal activity against the pathogen pythium ultimum . compositions and methods for treating or protecting plants susceptible to pythium ultimum damage , and pythium sp . “ group g ” damage in particular , are provided . such strains include , for example , the strains deposited in the international depository authority of canada under accession numbers idac 200704 - 01 , idac 200704 - 02 , idac 200704 - 03 , and idac 200704 - 04 .

Description:
we have found that pythium diseases may be controlled by strains of the nitrogen - fixing bacteria rhizobium leguminosarum bv . viceae . in one study , fifty - six percent of strains of r . leguminosarum bv . viceae obtained from field pea and lentil nodules were found to improve emergence of sugar beet seedlings in soil artificially infested with the pathogen pythium sp . “ group g ” in indoor experiments . strains tested as seed treatments in diseased fields effectively controlled damping - off of sugar beet and field pea caused by pythium spp . the effectiveness of the rhizobium strains is similar to that of pseudomonas fluorescens migula lrc 708 , a biological control agent against pythium damping - off of sugar beet , field pea , canola , and safflower ( bardin et al . 2003 ). the biological control activity of the rhizobium strains is not host dependent , as they are effective agents for both legume ( pea ) and nonlegume ( sugar beet ) plants . according to the present invention , r . leguminosarum bv . viceae strains are utilized to control or alleviate pythium infection on crops . rhizobium leguminosarum bv . viceae strains are useful for promoting plant health by protecting inoculated seeds from attack by pythium ultimum , and pythium sp . “ group g ” in particular , thereby reducing incidence of damping - off diseases . the bacteria rhizobium leguminosarum bv . viceae comprising the active agent of the invention may be isolated from root nodules of host legumes such as pea and lentil . the bacteria are collected from root nodules which are removed from the plants and crushed . the nodule contents are plated on an appropriate medium to support the growth of the bacteria . the bacteria are cultured under conditions favoring the growth thereof . conditions for culturing rhizobium leguminosarum strains are known to those skilled in the art , and are exemplified in the examples which follow . colonies are isolated from the culture . the antagonistic activity of isolates against pythium ultimum may be determined using the dual - culture technique . each bacterial strain is streaked on a medium which will support the growth of pythium ultimum , e . g ., tryptone yeast extract agar near the edge of a petri dish ( 9 cm diameter ). after incubation for one day at room temperature , a mycelial plug ( 0 . 6 mm diameter ) of pythium ultimum from a 48 - hour culture grown on potato dextrose agar is placed in the center of each dish containing the bacterial streaks . the plates are incubated for five days at room temperature ( 20 ± 2 ° c .) and the inhibitory effect of each bacterial strain is determined by measuring the inhibition zone of mycelial growth . the inhibitory effect is scored as positive where the pythium growth stops on or before the bacterial streak line . the bacteria may be utilized in the form of cultures of bacteria , such as a suspension in a whole broth culture , to prepare appropriate compositions for ground treatment , plant treatment , soil and / or growing media treatment , or seed treatment . the compositions containing the bacteria as the sole active ingredient , or as a combination with one or more other active ingredients , are prepared in known manner , such as by using standard fermentation methods , processes and equipment , followed by homogeneously mixing and / or grinding the active ingredients with extenders , growth media ingredients ( for example such as nutrients , stabilizers , buffering systems , plant growth hormones , and ph adjustment ingredients ) and liquid or dry organic or inorganic carriers . suitable carriers include sterilized and sanitized liquid carriers ; pre - sterilized ( irradiated or steam sterilized ) and non - sterilized peat powders ; granulated , spheronized or pelletized peat , clay ; and other extenders , filler pigments or minerals . other carriers for granular formation include talc , gypsum , kaolin , attapulgite , montmorillonite , bentonite , wood flour , ground corn cob grits , starch , cellulose , and bran . the formulations can also contain additives such as adhesives , stickers , binders , polymers and other adjuvants applicable to agricultural or horticultural applications . stickers or binders may comprise , for example , ethylene glycol , mineral oil , polypropylene glycol , polyvinylacetate , lignosulfonate , polyvinyl alcohol , polyvinylpyrrolidone , graphite , gum arabic , methyl cellulose , and sucrose . the compositions of this invention can be formulated in powder or granular form by mixing together all the components , including any carrier and / or other additive ( s ) which may be utilized until a homogeneous mixture is formed . a sticker , if employed , may then be added and the entire mass mixed again until it has become essentially uniform in composition . the composition may or may not be formulated in a pre - sterilized carrier system . the optimum concentration of rhizobium leguminosarum bv . viceae employed in the compositions of the invention for a particular application can be readily determined by those skilled in the art . in general , the concentration of bacteria can range from about 0 . 001 to about 1 %, preferably from about 0 . 01 to about 0 . 5 %, more preferably from about 0 . 05 to about 0 . 1 %, by weight . in the case of a liquid formulation , an aqueous liquid nutrient medium may be utilized , optionally comprising adjuvants such as stickers , stabilizers and colorants . the composition of the invention may contain , as additional active agents , cells , spores or propagules of other biological control agents , one or more chemical fungicides , or one or more other pesticidal materials , such as insecticides . the chemical fungicide may be selected on the basis of its activity against pythium spp ., or may be selected on the basis of activity against other fungal pathogens . the method of the invention comprises applying to plants an antifungal effective amount of a composition containing rhizobium leguminosarum bv . viceae . the composition is most advantageously applied to roots or seeds . the composition may also be utilized as ground treatments in fields or greenhouses . they may be applied to soil surrounding plants , or applied to soil into which seeds or seedlings are planted . the compositions are applied by methods which include , for example , seed treatments , spray applications , in - furrow applications , soil and growing media inoculation , application through irrigation system , and the like . the compositions can be applied as stand alone or with the standard chemical treatments to control pythium . when employed as a seed dressing , the amount of composition is applied such that the seed is coated with a concentration of bacteria adequate to provide protection against pythium spp . the actual amount to utilize depends on the nature and size of the seed , the amount of the protection desired , the local soil conditions , and other factors which may be taken into account in selecting the appropriate dosage of bacteria . appropriate application rates of bacteria in terms of colony forming units ( cfu ) per seed are as follows : large seeded crops — from about 10 4 to about 10 8 for legumes , and from about 10 6 to about 10 7 for non - legumes ; medium size - seeded crops — from about 10 3 to about 10 7 for legumes , and from about 10 5 to about 10 6 for non - legumes ; small seeded crops — from about 10 2 to about 10 6 for legumes , and from about 10 4 to about 10 5 for non - legumes . greater concentrations of bacteria may be applied . seeds may be bacterized according to the present invention by steeping the seeds in a suspension of bacteria for an appropriate time , e . g . one hour . according to one such technique , bacterial slurries are prepared by adding 3 ml of 1 % methyl cellulose to tryptone yeast medium plates on which the culture is grown , and gently scraping the culture off the plate . for seed treatment , seeds are soaked for , e . g ., 20 minutes in the bacterial slurry . the bacterial concentration in the slurry may be determined by plating serial dilutions on tryptone yeast medium ( beringer , 1974 ). according to one embodiment , an inoculant composition may be prepared for application to seeds , using ground peat . methods for the preparation of inoculant compositions of rhizobium sp . for inoculation of crops , e . g ., legumes , to increase nitrogen fixation are known . see , e . g ., u . s . pat . no . 5 , 484 , 464 , the entire disclosure of which is incorporated herein by reference . one such inoculant composition is prepared from sterilized powdered peat with a moisture content of 6 - 20 %, with or without a sticker . using aseptic techniques , a suspension of rhizobium leguminosarum bv . viceae is added to the peat at a rate of from about 10 5 to about 10 8 colony forming units of bacteria per gram of peat . the composition may be utilized to protect any crop which is susceptible to infection and damage by pythium ultimum , and pythium sp . “ group g ” in particular . such crop species include , for example , sugar beet ( beta vulgaris l . ), field pea ( pisum sativum l . ), lentil ( lens spp . ), safflower ( carthamus tinctorius l . ), canola ( brassica rapa l . and brassica napus l . ), chickpea ( cicer spp . ), sunflower ( helianthus spp . ), alfalfa ( medicago spp . ), soybean ( glycine spp . ), and field bean ( vicia faba ). in the following examples , the viable counts of bacterial agents in slurries and on seeds were expressed as mean cfu ± se . shoot dry mass and emergence data of both indoor and field experiments were analyzed statistically using the statistic analysis software package version 6 . 0 . 9 ( examples 1 - 6 ) or version 8 . 2 ( example 7 ) ( sas institute inc ., cary , n . c .). analysis of variance was done using the general linear model procedure . differences between treatments were analyzed using fisher &# 39 ; s least significant difference ( lsd ) test . all analyses were performed at the p = 0 . 05 level . strains of r . leguminosarum bv . viceae were isolated from root nodules of field pea and lentil grown in southern alberta , canada , as follows . roots from two plants per crop were washed in water to remove soil particles . the nodules were excised , surface sterilized in 2 % sodium hypochlorite for 1 min , washed eight times in sterile distilled water , and crushed with a sterile spatula in 200 μl sterile water . the nodule contents were plated on tryptone - yeast extract medium ( ty ; beringer 1974 ) containing 1 . 5 % agar ( difco , detroit , mich .). following incubation for 3 - 4 days at room temperature ( 20 ± 2 ° c . ), a colony from each plate was purified by three successive single colony isolations . eighteen strains of r . leguminosarum bv . viceae were isolated in this manner . ten strains were isolated from field pea root nodules and eight from lentil root nodules . of these strains , 8 showed no potential for control of pythium damping - off of sugar beet in preliminary indoor experiments and were not tested further . the identities of the 10 remaining strains , 8 from pea and 2 from lentil ( table 1 ), were confirmed by performing plant nodulation experiments ( see below ) and by streaking the bacteria on luria - bertani ( l b , miller 1972 ) agar . the strains did not grow on lb , which is consistent with the fact that r . leguminosarum is sensitive to the high salt concentration contained in this media . the ability of the ten pythium - antagonizing rhizobium isolates to form nitrogen - fixing nodules on pea and lentil plants was determined in a nitrogen - free medium . seeds were surface sterilized for 5 min in 50 % aqueous sodium hypochlorite , washed 8 - 10 times with sterile distilled water , and germinated for 2 days in the dark on water agar ( 1 . 5 %) in petri dishes . six seeds were planted in each sterile leonard jar assembly ( leonard 1943 ), containing a mixture of quartz sand and vermiculite ( 1 : 1 ; v / v ) saturated with nitrogen - free jensen &# 39 ; s nutrient solution ( vincent 1970 ). two days after planting the seeds , each jar was inoculated with 10 ml of an aqueous bacterial suspension ( 10 7 - 10 8 cfu / 10 ml ) of rhizobium or with 10 ml water for the uninoculated control . each treatment was performed in duplicate . the experiment was repeated once . the plants were kept in a growth cabinet in a 16 h light ( 20 ° c . ): 8 h dark ( 15 ° c .) cycle . they were watered with sterile distilled water as required . lentil and pea plants were collected 26 and 27 days after inoculation , respectively . the shoots of the plants were excised , dried in a 60 ° c . oven for 5 days , and weighed to determine nodulation efficacy . plants inoculated with each of the ten pythium - antagonizing rhizobium strains were green and healthy compared with the brown and stunted plants of the uninoculated control . there were pinkish nodules formed on the roots of plants inoculated with the strains , while no nodules developed on the roots of uninoculated plants . in addition , the dry shoot masses of the inoculated plants were significantly ( p & lt ; 0 . 05 ) greater than those of uninoculated plants ( table 1 ). strain r12 was effective in establishing a beneficial symbiotic interaction with both lentil and pea plants ( table 1 ). the antagonistic activity of the ten remaining r . leguminosarum bv . viceae strains against pythium sp . “ group g ” strain lrc 2105 ( huang et al . 1992 ) was determined by streaking a rhizobium strain 4 cm away from a potato dextrose agar ( pda ) plug colonized by pythium on ty agar plates ( dual culture technique ). after incubation at room temperature for 5 days , the inhibitory activity of the rhizobium strain was determined by measuring the zone of mycelial growth inhibition around the bacterial streak . three ratings were used : −, no inhibition zone and growth of pythium over the bacterial streak ; +, no inhibition zone , but no growth of pythium on the bacteria streak ; and ++, 1 - 5 mm inhibition zone . there were three replicates for each treatment and the experiment was repeated once . strain r5 was rated as ++. it was the only strain showing antagonistic effects to pythium sp . “ group g ”, with formation of a small zone of inhibition 2 mm in size . the other 9 strains did not exhibit zones of inhibition but were able to prevent colonization of the bacterial streak by the pathogen and were therefore rated as + ( slight inhibition ). protease production was determined by incubating colonies of r . leguminosarum bv . viceae on skim milk agar plates ( dunne et al . 1997 ) for 5 days at room temperature ( 20 ± 2 ° c .). protease activity was compared with the protease positive strain , pseudomonas fluorescens migula lrc 708 , which degrades casein and causes clearing of the skim milk agar plate ( bardin et al . 2003 ). there were three replicates for each treatment and the experiment was repeated once . unlike strain p . fluorescens 708 , none of the rhizobium strains tested showed protease activity , as they failed to produce clearing zones around the colonies when plated on the skim milk agar plates . thus , production of extracellular proteases is not the mechanism of action of the rhizobium strains . the strains of r . leguminosarum bv . viceae were further tested as seed treatments for control of pythium damping - off of sugar beet in nonsterile soil . bacterial cultures were grown on ty agar in petri dishes ( 5 . 5 cm in diameter ) for 48 hours at room temperature . the bacterial culture was resuspended in 3 ml of 1 % methyl cellulose ( mc ) ( aldrich chemical , milwaukee , wis .) by scraping the agar surface gently with a spatula . this resulted in bacterial slurries with a concentration averaging 3 . 9 × 10 9 ± 0 . 5 × 10 9 ( mean ± se ) cfu / ml . sugar beet ( beta vulgaris ‘ hm bergen ’) ( novartis seeds — hilleshög , longmont , colo .) seeds were soaked for 20 minutes in the mc - bacterial slurry and were seeded directly into soil artificially infested with pythium sp . “ group g ” strain lrc 2105 . the soil consisted of 3 parts topsoil ( bzdell soil service , lethbridge , alberta . ), 1 part sand ( tollestrup construction , lethbridge , alberta ), and 1 part peat moss ( premier horticulture , red hill , pa .). the pythium inoculant was prepared in pans containing a sterile mixture of 150 g wheat bran ( ellison milling , lethbridge , alberta ), 150 g corn meal ( mccormick , london , ontario ), and 300 ml distilled water . twenty plugs ( 8 mm in diameter ) of a 48 - hour - old pda culture of pythium sp . “ group g ” were placed in each pan . after incubation for 2 weeks at room temperature in the dark , the wheat bran - corn meal mix was completely colonized by the pathogen . the pythium inoculum was air - dried at room temperature for 4 days and ground using a thomas - wiley model 4 laboratory mill ( thomas scientific , philadelphia , pa .) equipped with a 1 - mm mesh screen . the soil , artificially infested with pythium sp . “ group g ” at a concentration of 2 g inoculum / kg soil , was used to fill root trainers ( spencer - lemaire industries , edmonton , alta . ), each containing 17 books of six cells per book . one sugar beet seed was planted per root trainer cell at a depth of 1 . 5 cm . uninoculated seeds were also planted in non - infested soil . the root trainers were soaked in a water - filled tray until the soil was saturated by capillary action , and were then placed in propagator trays ( the stewart company , croydon , surrey , uk ) to create a high - moisture environment . the propagator trays were kept in a growth chamber in a 16 hour light ( 20 ° c . ): 8 hour dark ( 15 ° c .) cycle . in each experiment there were three replicates per treatment and 18 seeds per replicate . the treatments were arranged in a completely randomized design . seedling emergence was recorded 14 days after planting , and data from bacterial seed treatments were compared with the uninoculated control . each set of experiments was repeated twice . non - germinated seeds were collected , washed with sterile water , surface sterilized in 70 % ethanol for 2 min , and plated on pda in petri dishes . the fungi isolated from the seeds were purified on pda , and the genus of each fungus isolated was determined based on morphological characteristics . emergence of uncoated sugar beet seeds planted in the pythium - infested soil used in the indoor experiment was reduced by 37 % ( 21 % emergence ) compared with seeds planted in non - infested soil ( 58 % emergence ). pythium was reisolated from 65 % of the non - germinated seeds tested . despite the lack of clear antagonism against pythium sp . “ group g ” in the in vitro assays , seed treatment with the rhizobium strains significantly ( p & lt ; 0 . 05 ) increased emergence of sugar beet in soil artificially infested with pythium sp . “ group g ” compared with the untreated control ( table 2 ). the most effective strains for biological control of damping - off of sugar beet were r3 , r4 , r5 , r7 , r12 , r20 , and r21 ( table 2 ). the selected strains of r . leguminosarum bv . viceae ( r12 , r20 , and r21 ) effective against pythium damping - off of sugar beet in indoor experiments were tested for control of damping - off of sugar beet and field pea in fields naturally infested with pythium spp . at the lethbridge research centre , alberta . the efficacy of the rhizobium strains was compared with the biocontrol agent p . fluorescens 708 , which was shown to improve emergence of sugar beet , field pea , canola , and safflower in soil naturally infested with pythium spp . ( bardin et al . 2003 ). the seeds were coated with the bacterial slurry as described previously using 2 . 4 and 9 . 5 ml bacterial slurry / 100 seeds of sugar beet and field pea , respectively . the seeds were dried overnight at room temperature on a metallic mesh , which was placed on a paper towel to absorb the excess slurry . the number of bacteria coated onto the seeds was similar for the four bacterial strains , ranging from 1 . 4 × 10 6 ± 0 . 2 × 10 6 to 2 . 3 × 10 7 ± 0 . 2 × 10 7 cfu / seed for sugar beets and 3 . 0 × 10 7 ± 0 . 3 × 10 7 to 1 . 2 × 10 8 ± 0 . 4 × 10 8 cfu / seed for field peas . the coated seeds were then stored at 4 ° c . until planting . bacterial counts on the seeds were determined by vortexing five coated seeds in 5 ml of distilled sterile water for 30 seconds , and by plating serial dilutions on ty agar medium in petri dishes for rhizobium strains and on pda in petri dishes for p . fluorescens . each bacterial count was performed in duplicate , and bacterial determinations for each treatment were performed twice . the rhizobium - treated and untreated seeds were machine seeded into 0 . 9 m wide × 5 . 0 m long plots made of 4 rows of 100 seeds / row in a field naturally infested with pythium spp . the plots were trimmed to 3 . 5 m after all seedlings emerged . treatments were arranged in a randomized complete block design , with six replicates per treatment . the field experiments were performed twice , once in may and again in august 2001 in fairfield farm , lethbridge , alberta . seedling emergence was recorded 4 weeks after planting and was compared with the uninoculated and fungicide controls . the amount of thiram ™ for the fungicide - treated seeds was 90 g / 25 kg sugar beet seeds and 30 g / 25 kg field pea seeds . treatment of pea seeds with r . leguminosarum bv . viceae strain r12 or r20 caused a significant ( p & lt ; 0 . 05 ) increase in seedling emergence compared with the untreated control in the two field experiments ( table 3 ). the efficacy of the two rhizobium strains was similar to that of seed treatments with the rhizobacterium p . fluorescens 708 . rhizobium leguminosarum bv . viceae r21 significantly increased pea seedling emergence compared with the untreated control in the second ( august 2001 ) but not in the first ( may 2001 ) field experiment . the level of seedling emergence in the second field experiment was lower but not significantly ( p & gt ; 0 . 05 ) different from that of r . leguminosarum bv . viceae r20 and p . fluorescens 708 . none of the bacterial treatments were as effective as the fungicide thiram ™ for control of damping - off of field peas . in the sugar beet experiments conducted in may and august of 2001 , seed treatment with r . leguminosarum bv . viceae r12 , r20 , or r21 increased seedling emergence compared with the untreated control ( table 3 ). this increase was significant ( p & lt ; 0 . 05 ) in the august experiment . in both the may and august field experiments , the percent emergence of the rhizobium - treated seeds was not significantly different from that of seeds treated with p . fluorescens 708 . rhizobium leguminosarum bv . viceae r12 and r21 were as effective as the fungicide treatment for protection of sugar beet seedlings against pythium damping - off in the august field experiment , while p . fluorescens 708 was as effective as the fungicide treatment in the may field experiment ( table 3 ). the strains of rhizobium leguminosarum bv . viceae used for the study were 99a1 , r12 , r20 , and r21 . strain 99a1 was originated from the commercial pea inoculant produced by agrium , inc . calgary , alberta . bacterial cultures were grown on tryptone - yeast agar ( tya ) ( beringer , 1974 ) in petri dishes for 48 h at room temperature ( 20 ± 2 ° c .). the resulting colonies were suspended in 5 ml per dish of 1 % methyl cellulose ( sigma - aldrich , milwaukee , wis .) in sterile distilled water , and scraped gently with a spatula to obtain bacterial slurries . seeds of field pea cv . trapper and lentil cv . laird were soaked for 20 minutes in the slurries , spread on a metallic mesh sheet with paper towel underneath to absorb the excess slurry , and air - dried overnight under a fume hood . enumeration of bacteria coated onto seeds was done by placing 5 seeds in a test tube with 5 ml of sterile distilled water , vortexing for 30 sec , and plating serial dilutions on tya , 0 . 1 ml per 9 - cm dish . after incubation at room temperature for 3 days , bacterial colonies developed in each dish were counted . there were two replicates for each treatment . field experiments were conducted at the agriculture and agri - food canada research centre near lethbridge , alberta , canada , in a field naturally infested with pythium spp . ( predominantly pythium sp . ‘ group g ’). for the pea experiment , seeds were planted using a plot seeder on 28 may 2004 , in 4 - row plots with a row length of 5 m , a row spacing of 22 . 5 cm , and a plant spacing of 5 cm ( i . e . 20 seeds / m ). untreated seeds and fungicide - treated seeds ( thiram ™ at the rate of 30 g / 25 kg seed ) ( gustafson ; calgary , alberta , canada ) were used as controls . treatments were arranged in a randomized block design with 6 replicates . for the lentil experiment , seeds were planted on the same date and using the same parameters as for field pea . seedling emergence for each plot was determined . the number of healthy seedlings and the number of wilted seedlings were counted in the middle 3 m of each row , and the percent loss due to pre - emergent and post - emergent damping - off were calculated , as well as the final stand establishment . the causal agent of seedling death was determined by collecting 10 non - emerged seedlings and all of the wilted seedlings from each plot , washing in running water , surface sterilizing in 70 % ethanol for 90 sec , incubating on potato dextrose agar ( pda ) in petri dishes at room temperature for 7 days , and examining the organisms derived from each sample . results of reisolation of diseased seedlings were used to calculate the incidence of damping - off due to pythium spp . for each plot . seedling height for each plot was determined ( 6 - node stage for peas ; 5 - node stage for lentils ). for each row , ten seedlings were randomly selected and the distance from the first node to the terminal branch of each seedling was measured . differences between treatments for incidence of damping - off , seedling emergence and seedling height data were analyzed for statistical significance using analysis of variance ( anova ) and means of treatments for each set of data were separated using duncan &# 39 ; s multiple range test at the p = 0 . 05 level . all statistical analyses were done using sas statistical analysis software , version 8 . 2 ( sas institute inc ., cary , n . c . 2001 ). the results are set forth in tables 4 - 7 . 1 distance from the first node to the terminal branch ; measured at the 6 - node stage ( 4 weeks after planting ). based on random selection of 10 seedlings per row , 4 rows per plot . 2 means within each column followed by the same letter are not significantly different ( duncan &# 39 ; s multiple range test ; p & gt ; 0 . 05 ). 1 distance from the first node to the terminal branch ; measured at the 5 - node stage ( 4 weeks after planting ). based on random selection of 10 seedlings per row , 4 rows per plot . 2 means within each column followed by the same letter are not significantly different ( duncan &# 39 ; s multiple range test ; p & gt ; 0 . 05 ). enumeration of bacteria coated onto seeds revealed similar numbers of bacteria per seed for all four strains of r . leguminosarum bv . viceae , for both field pea and lentil . the number of colony - forming units ( cfu ) per seed ranged from 2 . 3 × 10 5 to 2 . 9 × 10 5 for pea , and from 2 . 2 × 10 5 to 5 . 1 × 10 5 for lentil . reisolation of diseased pea seedlings revealed that 84 % of the seedlings killed by pre - and post - emergent damping - off were infected with pythium spp ., whereas the remaining seedlings were colonized by fusarium spp . treatment of pea seeds with r20 , r21 or thiram ™ significantly ( p & lt ; 0 . 05 ) reduced pre - emergent damping - off compared to the untreated control ( table 4 ). the incidences of pre - emergent damping - off for the treatments of r20 , r21 and thiram ™ were 51 . 6 %, 28 . 5 % and 20 . 3 %, respectively , compared to 62 . 0 % for the untreated control . there was no significant difference in incidence of pre - emergent damping - off between the treatments of r21 and thiram ™. damping - off losses of pea due to pythium spp . alone followed a similar trend , ranging from 16 . 7 % in the fungicide treatment and 23 . 7 % in the treatment of r21 , to 55 . 4 % in the untreated control ( table 4 ). the height of pea plants arising from seed treated with r12 , r21 or thiram ™ was significantly ( p & lt ; 0 . 05 ) greater than for plants arising from untreated seed ( table 5 ). seedling height for the treatments of r12 and r21 was 12 . 8 cm for both rhizobium strains , compared to 14 . 6 cm for the treatment of thiram ™, and 11 . 1 cm for the untreated control . for the lentil experiment , results of plating of diseased seedlings showed that 68 % were infected with pythium spp ., 22 % were infected with botrytis cinerea , and the remainder was colonized by fusarium spp . treatment of lentil seeds with r20 , r12 or thiram ™ significantly ( p & lt ; 0 . 05 ) reduced incidence of pre - emergent damping - off compared to the untreated control ( table 6 ). the disease incidences for the treatments of r20 , r12 and thiram ™ were 43 . 1 %, 39 . 9 % and 34 . 2 %, respectively , compared to 50 . 6 % for the untreated control . incidence of damping - off of lentil due to pythium spp . alone followed the same trend , ranging from 22 . 8 % in the fungicide treatment and 27 . 8 % in the treatment of r12 , to 38 . 1 % in the untreated control ( table 6 ). no significant differences in seedling height of lentil were detected among the treatments ( table 7 ). among the four strains of r . leguminosarum bv . viceae tested , strains r20 and r21 from pea were most effective for control of damping - off of pea ( table 4 ), whereas the strain r12 from lentil was most effective for control of damping - off of lentil ( table 6 ). the study on pea also suggests that the strains may have a growth promoting effect on seedlings , as seen in the case of increased height of pea seedlings for the treatments of r12 and r21 ( table 5 ). all references discussed herein are incorporated by reference . one skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned , as well as those inherent therein . the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and , accordingly , reference should be made to the appended claims , rather than to the foregoing specification , as indicating the scope of the invention . bardin , s . d ., and huang , h . c . 2001 . survey of damping - off diseases of sugar beet in southern alberta in 2000 . can . plant dis . surv . 1 : 136 - 137 . bardin , s . d ., huang , h . c ., liu , l ., and yanke , l . j . 2003 . control , by microbial seed treatment , of damping - off caused by pythium sp . on canola , safflower , dry pea and sugar beet . can . j . plant pathol . 25 : 268 - 275 . beringer , j . e . 1974 . r - factor transfer in rhizobium leguminosarum . j . gen . microbiol . 84 : 188 - 198 . brockwell , j ., bottomly , p . j ., and thies , j . e . 1995 . manipulation of rhizobia microflora for improving legumes productivity and soil fertility : a critical assessment . plant soil , 174 : 143 - 180 . dileep kumar , b . s ., berggren , i ., and martensson , a . m . 2001 . potential for improving pea production by co - inoculation with fluorescent pseudomonas and rhizobium . plant soil , 229 : 25 - 34 . drapeau , r ., fortin , j . a ., and gagnon , c . 1973 . antifungal activity of rhizobium . can . j . bot . 51 : 681 - 682 . dunne , c ., crowley , j . j ., moenne - loccoz , y ., dowling , d . n ., de bruijn , f . j ., and o &# 39 ; 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