interactions between salmonella enterica subspecies enterica serovar typhimurium and cowpea (vigna...

INTERACTIONS BETWEEN SALMONELLA ENTERICASUBSPECIES ENTERICA SEROVAR TYPHIMURIUM AND

COWPEA (VIGNA UNGUICULATA VARIETY SINENSIS) SEEDS,PLANTS AND PERSISTENCE IN HAY

BHOJ RAJ SINGH1, MUDIT CHANDRA, RAVIKANT AGARWAL andNAGRAJAN BABU

National Salmonella Center (Veterinary)Division of Bacteriology and Mycology

Indian Veterinary Research InstituteIzatnagar-243 122, India

Accepted for Publication September 18, 2006

ABSTRACT

Dynamics of persistence of Salmonella enterica subspecies entericaserovar Typhimurium (S. Typhimurium), a common food- and waterbornezoonotic serovar, on cowpea (Vigna unguiculata variety sinensis), a fodderand green vegetable plant, were studied. The findings revealed that S.Typhimurium not only reduced germination of cowpea seed (P < 0.01), butalso caused defects in sprouts (P < 0.02). S. Typhimurium inoculation onseeds before sowing had a more pronounced effect (P < 0.01; i.e., loss ingermination and appearance of defective sprouts) than sowing seeds in S.Typhimurium-inoculated soil. S. Typhimurium persisted in saplings andadult plants up to 45 days of plant age and up to 60 days in hay. Thecowpea plants grown in sterile Salmonella-free soil did not support coloni-zation of S. Typhimurium in different parts. A reduction in the population ofSalmonella appeared as early as on the fifth day and decreased with advanc-ing plant age. At 21 days of age, the cowpea plants had no Salmonella intheir aerial parts and were free of the pathogen within 3 h of inoculation.Salmonella persisted in stumps of the plants throughout the observation,irrespective of age of the plants at the time of inoculation. The studyrevealed the persistence and the phytopathogenic potential of Salmonella oncowpea.

1 Corresponding author. National Research Center on Equines, Sirsa Road, Hisar-125 001, India. TEL:0091-1662-275114, 276151 (O), +91-9813-011578 (R), +91-9813450133 (M); FAX: 0091-1662-276217. EMAIL: [email protected]

Journal of Food Safety 27 (2007) 169–187. All Rights Reserved.© 2007, The Author(s)Journal compilation © 2007, Blackwell Publishing

169

mailto:[email protected]

PRACTICAL APPLICATIONS

The results of the study will be useful in understanding the epidemiologyof salmonellosis, particularly as a foodborne pathogen, and the revelation ofthe phytopathogenic potential of Salmonella for cowpea opens a new vista forresearchers to understand the mechanism of pathogenesis of salmonellosis,knowledge of which may be applied for other plant pathogens. The resultsindicating that seedlings grown in Salmonella-free soil are resistant to invasionof Salmonella in their later life may be exploited for raising Salmonella-freeplants or vegetable products and may have impressive economic impact onagro-industry.

INTRODUCTION

Salmonellosis is endemic or hyperendemic in most parts of the world,affecting millions of people every year even in most developed countries.Every year, thousands of animals suffer from Salmonella infection, and mil-lions of livestock become carriers of Salmonella to act as a source of thepathogen to the human population and other animals. There are many sourcesof infection to animals, including environment, feeds and fodder (Wray andDavies 2000). Isolation of Salmonella from different feed ingredients of veg-etable origin is being increasingly reported (DEFRA 2004); consumption ofcontaminated feed results in either outbreaks of apparent disease or animalsbecoming carriers (Wray and Davies 2000; DEFRA 2004). Studies on pastureland grasses (Taylor and Burrow 1971; Kelly and Collins 1978), maize (Singhet al. 2004) and tomato plants (Guo et al. 2001, 2002) have shown that Sal-monella travels through and persists in plant tissues, and that pasture contami-nation may be the cause of disease outbreaks (Rasch and Richter 1956; Jeckand Hepper 1969). From the contaminated soil, Salmonella has been shown toreach the susceptible hosts via contaminated grasses, vegetables and sprouts(Clegg et al. 1983). Seeds have been reported to be the major source ofSalmonella in sprouts (NACMCF/FDA 1999). Enteric pathogens have beenshown to reside within the interiors of apples, lettuce and maize plants, andalfalfa and mung bean sprouts (Burnett et al. 2000; Gandhi et al. 2001;Kenney et al. 2001; Weissinger et al. 2001).

Recent studies (Yuemei et al. 2003) on the dynamics of SalmonellaTyphimurium and other enteropathogens on alfalfa and Lucerne (Medicagosativa and Medicago truncatula) plants and maize plants (Singh et al. 2004)demonstrated the rhizosphereic and endophytic colonization of enteropatho-gens to a variable extent depending on enteropathogenic strains. Cowpea(Vigna unguiculata variety sinensis), a multiseasonal legume crop, is native to

170 B.R. SINGH ET AL.

India and the Middle East. It is extensively grown as a hay crop and green-manure crop, and the beans (used as green vegetable) for human consumption.However, little is known about the effect of Salmonella on cowpea seeds andplants.

MATERIALS AND METHODS

Bacterial Culture and Preparation of the Inoculum

The inoculum was prepared from frozen stock of gentamicin-sensitive(MIC, 0.2 mg/mL) S. Typhimurium (E2391), an isolate from the spleen ofpoultry birds, which died in a maize grain-associated outbreak, by growing thepathogen in trypticase soy broth (TSB, Becton Dickinson and Co., Sparks,NV) as described earlier (Fett and Cooke 2003). Bacterial cells were harvestedby centrifugation (5,000 ¥ g, 15 min), and the cell pellet was suspended in0.01 M sodium phosphate buffer (PBS, pH 7.2). Colony-forming units (cfu) ofS. Typhimurium in the inoculum were determined by plating serially diluted(10-fold dilutions made in PBS) aliquots in triplicate on HEA (Hektoen entericagar, Becton Dickinson and Co.) plates. Each fresh inoculum was preparedfrom a single stock for inoculation of soil, water or seeds. For negative control,an equal amount of the similar inoculum was used after killing the pathogen byheating at 90C for 30 min. For checking the sterility of the heated preparation,1 mL of it was inoculated in 10 mL of fluid thioglycollate medium and incu-bated for 48 h at 37C to observe any growth.

Preparation of Pots for Sowing

Autoclaved plastic pots (AK Scientific Industries, Delhi, India) of 3.75-Lcapacity were filled with 3 kg of potting soil procured from the section ofHorticulture, Indian Veterinary Research Institute, Izatnagar. To sterilize thesoil, pots with soil were autoclaved at 121C for 30 min and then placed in apolypropylene tray (AK Scientific Industries). To inoculate the pot-soil with S.Typhimurium, the pots were irrigated with 500 mL of water containing9.8 ¥ 107 cfu of S. Typhimurium/mL 7 days before sowing. The control potswere irrigated with 500 mL of water containing an equal amount of heat-killedSalmonella as in the test. For postsowing inoculation, the water (500 mL/pot)containing 9.8 ¥ 107 cfu of S. Typhimurium/mL was applied 2 h after sowing.All the pots were kept in an airy room with a fiberglass roof to protect frominsects, pests and rainwater. Room air and soil were free of Salmonella, and itwas affirmed at monthly intervals through multiple site soil testing and airsampling for Salmonella (Edwards and Ewing 1986; Senior 1996). No arrange-ments were made to control humidity or temperature because the experimentwas carried out during the cowpea cropping season (June to September).

171INTERACTION OF SALMONELLA WITH COWPEA

Salmonella Count in Soil, Seed, Seedlings and Plants

Plants or their parts were harvested aseptically in individual sterile con-tainers using a separate set of sterilized forceps, spatula and scissors. Tengrams of seeds/seedlings/plant tissues were homogenized individually in100 mL of PBS with Ultra Turrax T-25 (IKA; Werke Gmbh and Co. KG,Berlin, Germany) homogenizer for 3 min. The cfu of S. Typhimurium inhomogenates were determined by plating 100 mL of serially diluted (in PBS)aliquots in triplicate on HEA plates (Becton Dickinson and Co.); suspectedcolonies were confirmed through plate agglutination test with specific (Ofactor 5 and H factor i and 2) serum (Edwards and Ewing 1986). Ten mil-liliter of homogenates was also inoculated into 90 mL of universal preen-richment broth (Becton Dickinson and Co.) and was incubated at 37C for16 h. Thereafter, a 1-mL aliquot was transferred to tetrathionate broth (TTB,Becton Dickinson and Co.) and 0.1 mL into 10 mL of Rappaport Vasilliadismedium (RV, Becton Dickinson and Co.), and was incubated at 37C for24 h. Then, the cultures were streaked on HEA and brilliant green agar(Becton Dickinson and Co.) plates and were incubated at 37C for 24 h fordetecting Salmonella colonies. Suspected colonies were confirmed as men-tioned earlier.

For determining S. Typhimurium in soil, 10 g of soil was suspended bywhirling in 100 mL of sterile distilled water, and then the suspension wasprocessed similar to plant tissue homogenate. Five presumptive colonieswere confirmed by agglutination with O factor 5 and H factor i and 2 spe-cific serum (Becton Dickinson and Co.) according to the manufacturer’sinstructions.

Evaluation of Effect of S. Typhimurium on Germination of Cowpea

Certified seed of cowpea cultivar UPC-607, purchased from a retail outletof GB Pant University of Agriculture and Technology, Pantnagar, Uttaranchal,were checked for the presence of Salmonella as described earlier by takingthree samples of 25 g each. The seeds were randomly divided in groups of 100seeds each. Treatments to seeds of different groups are summarized in Table 1.After various treatments, excess fluid was drained off and seeds were placedon six layers of wet cheesecloth placed on seed-germinating plates (AKScientific Industries) for 48 h in the dark at 30C. To determine the rate ofgermination, the number of seeds showing a visible radicle (root) and openingto the hypocotyls was counted.

Salmonella count was made before and after gentamicin treatment todetermine the endophytic and epiphytic population of Salmonella in germi-nating seeds using the method of Singh et al. (2005).


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Evaluation of Effect of S. Typhimurium on Cowpea Seeds Sown in Soil

The experiment was set with Salmonella-free cowpea seeds (C, D, E, F)as well as seeds inoculated with S. Typhimurium (A, B), by soaking in watercontaining 7.2 ¥ 106 cfu of S. Typhimurium/mL for 30 min and then washedtwice with sterilized distilled water as mentioned earlier. In each pot, 15 seedswere sown. Thirty-six pots were divided into groups A to F (Table 2); six potsin each group had either sterile (A, C, E) or nonsterile soil (B, D, F) and wereeither inoculated with Salmonella (C, D) or not inoculated with Salmonella (A,B, E, F). Observations were made for number of seeds germinated (on thethird, fifth and seventh days of sowing), height of saplings (on the fifth day)and count of S. Typhimurium in different parts (leaves, stem cut 5 cm abovebase, stump of 5 cm of basal stem) of plants (on the seventh day of sowing),and whole (cut 5 cm above base) plants (on the 7th, 14th, 21st, 28th, 35th and45th day of sowing) were harvested aseptically as mentioned earlier.

To count the endophytic S. Typhimurium, three plants from each groupwere cut at a height of 5 cm from the ground and dipped into 1,000 mL ofaqueous solution of gentamicin (200 mg/mL) for 30 min, leaving the cutsurface outside the solution (Singh et al. 2005). Thereafter, the plants weretaken out and the undipped portion was clipped off. The remainder of the plantwas washed twice with sterilized distilled water (1,000 mL) and was processedfor counting Salmonella. Similarly, the plants from the respective pots, but nottreated with gentamicin, were used as controls.

Evaluation of Effect of Age on Susceptibility of Cowpea Plants toS. Typhimurium Inoculation

To determine the number of Salmonella invading and surviving in differentparts of the cowpea plants at different stages of growth, each of the 24 pots withsterile soil was seeded with 15 Salmonella-free cowpea seeds and dividedequally into six groups (1–6). For inoculating pots with S. Typhimurium,500 mL of distilled water containing 7.2 ¥ 106 cfu of the pathogen/mL wereused for irrigation of groups 1, 2, 3, 4 and 5 pots on the 0, 5th, 10th, 15th and21st days of sowing, respectively. For irrigation, 500 mL of sterile distilledwater was used after 2 h of sowing and thereafter at a weekly interval. ForSalmonella counts in plants, three plants from each of the group were cut at aheight of 5 cm from the base. On the day of S. Typhimurium inoculation, theplants were cut at 1, 2, 3, 6, 12, 18 and 24 h and divided into three parts, namelythe basal (10 cm of stem), top leaves and branches with the rest of the stem. Onthe 5th, 10th and 21st days of Salmonella inoculation, the plants were cut andprocessed as whole for Salmonella count. On the 21st day of Salmonella inocu-lation, stumps of the plants were also pulled out and processed for Salmonellacount determined after homogenization of plant tissues as described earlier.


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Survival of Salmonella on Hay

To determine the survival of S. Typhimurium on hay of cowpea, 30 potswith sterile potting soil were sown with Salmonella-free cowpea seeds (five ineach). Half of the pots (A group) were irrigated with water containing S.Typhimurium (7.2 ¥ 106 cfu/mL) and the other half (B group) with steriledistilled water 2 h after sowing. Thereafter, the sterile distilled water was usedfor all irrigations. After 45 days of growth, plants were harvested and placed inseparate sterilized trays for 7 days to make hay in the greenhouse itself. GroupA hay (moisture content <16%) was stored at room temperature in dry contain-ers (sterilized polypropylene containers with airtight lids), while group B hay(tested Salmonella free) was drenched with distilled water containing S. Typh-imurium (7.2 ¥ 106 cfu/mL) and was again dried for 2 days at 37C to reduce themoisture content to <16%, and stored asA. Samples were taken for enumerationof S. Typhimurium on the 10th, 22nd, 30th, 40th, 50th and 60th days of harvest.

Statistical Analysis

The mean values, variance and SDs for seed germination, defectivesprouts, Salmonella counts in different parts of cowpea plants and at differenttimes for various treatments were calculated using results of replicates. Theresults from various treatments were compared with control using MicrosoftExcel-2000. Significance of effect of various treatments was determined usingthe Student’s t-test (Rickmers and Todd 1967).

RESULTS

The results of the study on the effect of S. Typhimurium on germinationof cowpea seeds allowed to sprout in germination plates are given in Figs. 1and 2. The findings revealed that only live S. Typhimurium significantly(P < 0.01) lowered the germination rate (Fig. 1), while killed (group A) S.Typhimurium had no effect on the germination. The germination was 97% inthe control group (A), while in group C inoculated with 7.2 ¥ 106 cfu of S.Typhimurium/mL, only 31% of seeds germinated. However, at a lower level(7.2 ¥ 104 cfu/mL) of S. Typhimurium inoculation, the germination of seedswas not significantly affected; it was 89.5%. Gentamicin treatment ofSalmonella-inoculated seeds after 4 h of inoculation resulted in a significant(P < 0.01) increase in germination (66.3%). However, gentamicin treatmentafter 48 h of S. Typhimurium inoculation had no significant effect on germi-nation as the germination was only 34.3% (Fig. 1). Gentamicin treatment ofseeds after 4 h of Salmonella inoculation also reduced the Salmonella popu-lation on seeds by about 5 log10 as compared with the untreated seeds, but


gentamicin treatment after 48 h of inoculation did not reduce the Salmonellapopulation significantly (Fig. 2). Although after gentamicin treatment thepopulation of Salmonella on contaminated seeds was drastically reduced (itremained only 1.16 to 1.18 log10 cfu/g instead of >5.0 log10 cfu/g in nontreatedgroup), sprouts developed from such seeds had considerably high(7.6 log10 cfu/g) Salmonella counts (Fig. 2). And although in all the groups theSalmonella count was more on nongerminated seeds than on germinated ones,the difference was significant (P < 0.05) in gentamicin-treated groups (Fig. 2).

The results of effect of S. Typhimurium on the germination of cowpeaseeds sown in soil are shown in Table 3. At the same level of Salmonellainoculation and after 3 days of sowing, inhibition of cowpea seed germinationwas significantly (P < 0.05) lower (80.3%) when the seeds were sown in sterilesoil (Table 3). The germination rate was still less (35.5%) when Salmonella-inoculated seeds were sown in nonsterile soil. On the other hand, whenSalmonella-free seeds were sown in Salmonella-contaminated soil, inhibitionof germination was more (27.8%) in sterile soil than in nonsterile soil (39.5%).Besides lowering the germination rate, S. Typhimurium also increased thegermination time (Table 3), and this difference became more evident when soilwas contaminated with Salmonella or Salmonella-inoculated seeds were sownin nonsterile soil. The germination time was more in nonsterile soil irrespec-tive of contamination with Salmonella. The germination rate of S.Typhimurium-inoculated seeds sown in Salmonella-free soil was 91.8% on the

9789.5

31

66.3 67.5

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0102030405060708090

100

A B C D E F

Treatment groups of cowpea seeds

% g

erm

inat

ion

FIG. 1. SPROUTING OF COWPEA (UPC-607) SEEDS IN THE PRESENCE OF SALMONELLAENTERICA SUBSPECIES ENTERICA SEROVAR TYPHIMURIUM

A, seeds soaked in sterilized distilled water containing heat-killed Salmonella (7.2 ¥ 104 cfu/mL ofwater); B, seeds soaked in water inoculated with Salmonella (7.2 ¥ 104 cfu/mLof water); C, seeds soakedin water inoculated with Salmonella (7.2 ¥ 106 cfu/mL of water); D, seeds soaked in water inoculatedwith Salmonella (7.2 ¥ 106 cfu mL of water) and then treated with gentamicin (200 mg/mL) after 4 h ofsoaking; E, seeds soaked in water inoculated with Salmonella (7.2 ¥ 106 cfu/mL of water) and treatedwith gentamicin (200 mg/mL) after 4 and 24 h of soaking; F, seeds soaked in water inoculated with

Salmonella (7.2 ¥ 106 cfu/mL of water) and treated with gentamicin (200 mg/mL) after 48 h.


seventh day of sowing, which was significantly (P < 0.01) higher than theSalmonella-free seeds sown in Salmonella-contaminated soil (66.0%). In non-sterile soil, initially the germination of Salmonella-inoculated and Salmonella-free seeds did not differ much (Table 3); it became evident only after 5 days ofsowing. The germination of Salmonella-inoculated (A) and Salmonella-freeseeds (E) sown in sterile soil did not differ much even after 7 days of obser-vation (Table 3). On the seventh day of sowing, significant inhibition ofcowpea seed germination was observed only in group C, i.e., Salmonella-freeseeds sown in Salmonella-contaminated sterile soil.

The percentage of defective saplings was the maximum when S. Typh-imurium was present either in soil or on seed. Appearance of surface roots andkinked stem were the major defects in saplings in the presence of S. Typhimu-rium. The maximum number of defective saplings (33.8%) was observed in Cgroup, i.e., cowpea seeds sown in sterilized soil inoculated with Salmonellafollowed by that in B group (22.3%), Salmonella-inoculated seeds sown inSalmonella-free nonsterile soil.

0123456789

10

A B C D E FTreatment groups of cowpea seeds

Log

10 c

fu o

f S

alm

onel

la g

m-1

After 4 hours of inoculation Salmonella count in sproutsSalmonella count in non-germinated seeds

FIG. 2. SALMONELLA ENTERICA SUBSPECIES ENTERICA SEROVAR TYPHIMURIUMCOUNTS IN COWPEA (UPC-607) SPROUTS AND IN NONSPROUTED SEEDS UNDER

DIFFERENT CONDITIONS OF SPROUTINGA, seeds soaked in sterilized water; B, seeds soaked in water inoculated with Salmonella(7.2 ¥ 104 cfu/mL of water); C, seeds soaked in water inoculated with Salmonella (7.2 ¥ 106 cfu/mL ofwater); D, seeds soaked in water inoculated with Salmonella (7.2 ¥ 106 cfu/mL of water) and thentreated with gentamicin (200 mg/mL) after 4 h of soaking; E, seeds soaked in water inoculated withSalmonella (7.2 ¥ 106 cfu/mL of water) and then treated with gentamicin (200 mg/mL) after 4 h ofsoaking and then after 48 h; F, seeds soaked in water inoculated with Salmonella (7.2 ¥ 106 cfu/mL of

water) and then treated with gentamicin (200 mg/mL) after 48 h.


TAB

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Early growth of cowpea saplings did not differ much among differentgroups except in A group (Salmonella-inoculated seeds sown in sterile soil), inwhich the growth was significantly faster. Saplings grown in Salmonella-inoculated sterilized soil (C) or from Salmonella-inoculated seeds sown innonsterile soil had the minimum average growth, 8.5 and 8.8 cm, respectively,which was significantly less than in control (E and F) groups.

S. Typhimurium persisted up to 45 days in all parts of the plants growneither from the Salmonella-inoculated seeds or in Salmonella-contaminatedsoil. The Salmonella population in plants grown in presterilized soil wassignificantly higher (P < 0.05) than in plants grown in nonsterilized soil(Table 3). On the 14th day of plant life, gentamicin treatment of plants reducedthe total Salmonella count in plants by 4–16% in groups B, C and D, whilegentamicin treatment of plants grown from S. Typhimurium-inoculated seedsin sterile soil (group A), reduced the count about 2 log10, i.e., >98% of S.Typhimurium got killed.

Observations on Salmonella invasion in plant tissues revealed that inocu-lation of soil with Salmonella on the day of sowing and on the fifth day ofsowing resulted in dissemination of Salmonella to all parts of the plants. Theorganism could be isolated even after 21 days of observation. In plants of thepots of groups 3, 4 and 5 inoculated with S. Typhimurium after 10, 15 and 21days of sowing, the pathogen could be detected up to 12, 6 and 3 h, respec-tively after irrigation with Salmonella-contaminated water, and the pathogencould not be recovered from aerial parts of plants after 15 h (Table 4). S.Typhimurium persisted in cowpea plants in roots and stumps, irrespective ofthe age of the plants at the time of contamination of soil with the pathogen.S. Typhimurium could be detected in aerial parts of the plants of all fourgroups (2–5) within an hour of irrigation with the Salmonella-contaminatedwater.

On hay, S. Typhimurium persisted throughout the observation period of60 days, irrespective of its mode of entry onto cowpea plants (Fig. 3). Hayfrom the plants grown in Salmonella-contaminated soil had 4.18 log10 cfu of S.Typhimurium/g. On the 10th day of haymaking, S. Typhimurium populationwas significantly (P < 0.01) less on hay made from plants grown in S.Typhimurium-contaminated soil than hay contaminated with Salmonella afterharvest; however, from the 20th day onward, the difference in population ofthe pathogen on the two types of hay became insignificant.

DISCUSSION

With the demand and popularity of organic foods, including vegetables,milk, meat and other animal products, soil fertilization with compost as an


alternative to synthetic fertilizers has increased. Besides compost, sewageis the major source of organic matter to fertilize the soil, and it is beingcommonly used in all vegetable-growing regions in India, particularly nearmetropolitan cities. Moreover, an open system of defecation in vegetable fields

TABLE 4.EFFECT OF AGE OF COWPEA (VIGNA UNGUICULATA VARIETY SINENSIS) PLANTS ON

THEIR SUSCEPTIBILITY TO SALMONELLA TYPHIMURIUM

Time afterirrigation

Parts ofthe plant

Salmonella count (cfu/g in log10) in plants irrigated with 500 mLSalmonella (7.2 ¥ 106 cfu/mL) containing water at the age of

0 day* 5 days 10 days 15 days 21 days

1 h Top leaves 5.56 (0.05) 3.19 (0.19) AE (NC) AE (NC) 0.0 (0.00)Stem† 4.51 (0.17) 3.70 (0.17) 3.36 (0.22) 3.39 (0.11) AE (NC)Branches‡ 4.54 (0.04) 3.54 (0.09) 2.12 (0.08) 3.36 (0.10) AE (NC)

3 h Top leaves 4.42 (0.07) 3.22 (0.07) AE (NC) AE (NC) 0.0 (0.00)Stem† 3.83 (0.22) 3.16 (0.08) 3.13 (0.07) 3.14 (0.20) AE (NC)Branches‡ 3.89 (0.04) 3.21 (0.05) 2.80 (0.09) AE (NC) 0.0 (0.00)

6 h Top leaves 3.77 (0.10) 3.03 (0.01) 2.35 (0.27) 0.0 (0.00) 0.0 (0.00)Stem† 2.74 (0.25) 2.05 (0.8) AE (NC) AE (NC) 0.0 (0.00)Branches‡ 3.13 (0.14) 2.24 (0.09) 2.09 (0.28) 0.0 (0.00) 0.0 (0.00)

12 h Top leaves 3.62 (0.12) 2.91 (0.07) AE (NC) 0.0 (0.00) 0.0 (0.00)Stem† 2.40 (0.39) 1.06 (1.24) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Branches‡ 2.83 (0.13) 1.47 (0.98) AE (NC) 0.0 (0.00) 0.0 (0.00)

15 h Top leaves 3.39 (0.14) 2.82 (0.09) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Stem† 2.13 (0.07) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Branches‡ 2.09 (0.12) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

18 h Top leaves 3.11(0.04) 2.27 (0.13) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Stem† AE (NC) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Branches‡ AE (NC) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

24 h Top leaves 2.90 (0.06) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Stem† AE (NC) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Branches‡ AE (NC) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

5 days Whole plant§ 2.13 (0.28) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Whole plant¶ 2.80 (0.15) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

21 days Whole plant§ 2.57 (0.38) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Whole plant¶ 3.69 (0.20) AE (NC) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)Stumps** 4.49 (0.11) 3.88 (0.06) 2.64 (0.23) 2.83 (0.09) 2.48 (0.04)Stumps** 4.52 (0.08) 4.26 (0.19) 3.74 (0.16) 3.69 (0.18) 3.35 (0.09)

Figures in parentheses are the SDs of means of different observations.* Count was made on the fifth day of sowing.† Ten centimeters of stem from root side leaving 5 m stump.‡ Stem and branches above 15 cm of plant height.§ Plants harvested at 5 cm height from soil 2 h before irrigation.¶ Plants harvested at 5 cm height from soil 1 h after irrigation.** Five centimeter of plant stems left in soil after harvesting; AE, Salmonella could be detected after

enrichment for 18–24 h (detection limit �1 cfu/g).NC, SDs were not calculated.


(common in most of the developing counties) may lead to heavy contamina-tion of the soil, particularly at the defecation spot, and plants growing on thatparticular spot may be heavily infected with the pathogen. Sewage (Clegget al. 1983) and composts (Smith et al. 1982) have been reported to be thesources of Salmonella and other zoonotic pathogens. It is often believed thatanimal and human pathogens persist on plants without harming them. Earlierstudies suggested that Salmonella might enter into plants through abrasion onbark tissues, contamination of flowers and at early stages of development offruits (Guo et al. 2002). Multiplication of Salmonella in different parts ofalfalfa plants (Yuemei et al. 2003) has been reported recently. However, littleis known about the fate of Salmonella on cowpea, a common leguminousgreen fodder and hay crop.

Observation revealed that only live S. Typhimurium reduced the germi-nation of cowpea seeds (P < 0.01) above a certain threshold level, which is incontrast to earlier studies reporting no effect of Salmonella on germination ofalfalfa seeds (Fett and Cooke 2003). It may be either a result of the differencein phytopathogenic potential of S. Typhimurium strain or the susceptibilityof cowpea cultivar used in the study. Difference in invasive ability of S.Typhimurium in different germinating seeds has been reported earlier too(Jablasone et al. 2005). However, it is significant, as there may be otherSalmonella strains which may reduce germination of other cultivars of cowpeaand probably of other crops too, and needs further studies.

Gentamicin treatment of seeds after 4 h of inoculation with S. Typhimu-rium reduced the population of the pathogen by about 4 to 5 log10, indicatingthat the majority of Salmonella remained on the seed surface in the first 4 h of

0

1

2

3

4

5

6

0 10 20 30 40 50 60

Days after haymaking

Log

10 o

f av

erag

e C

FU

s gm

-1 o

f ha

y

A B

FIG. 3. SURVIVAL OF SALMONELLA ENTERICA SUBSPECIES ENTERICA SEROVARTYPHIMURIUM IN COWPEA (VIGNA UNGUICULATA VARIETY SINENSIS) HAY

A, Hay from plants harvested from pots inoculated with Salmonella at the time of sowing.B, Hay from Salmonella-free plants and then hay was inoculated with Salmonella.


inoculation. After gentamicin treatment, the remaining 1.18 log10 cfu ofSalmonella/g of seed multiplied to the extent of 7.6 log10 cfu of Salmonella/gon germinated and 9.16 log10 cfu of Salmonella/g on nongerminated seeds.Thus, the observations reflect that on invading into seeds, S. Typhimuriumgrew inside tissues of germinating seed and baby plants. These observationsare in concurrence with earlier reports on multiplication of Escherichia coliand Salmonella in germinating alfalfa (Lang et al. 2000; Yuemei et al. 2003),lettuce and radish seedlings (Jablasone et al. 2005).

The reduction of epiphytic (on the surface of cowpea seed) S. Typhimu-rium after gentamicin treatment significantly (P < 0.01) improved the germi-nation rate of S. Typhimurium-inoculated seeds. The observation revealed thatnot only endophytic (those that invaded the seed) but also epiphytic Salmo-nella have a role in inhibition of germination. It appears that S. Typhimuriumcells (1.18 log10 cfu/g) that survived gentamicin treatment might have inter-nalized in cowpea seeds and grew to sufficient numbers (9.2 log10 cfu/g) toinhibit the germination. Similar observations of internalization of S. Typhimu-rium in lettuce and radish seedlings grown from contaminated seeds have beenreported recently (Jablasone et al. 2005). Nongerminating cowpea seeds hadsignificantly (P < 0.05) higher numbers of S. Typhimurium than germinatedsprouts, particularly in the gentamicin-treated group (Fig. 2), indicating adefinite role of high load of Salmonella or its product in inhibiting the germi-nation of cowpea seeds.

Comparison between germination of cowpea seeds sown in soil and insprouting plates revealed that soil gave some protection against S. Typhimu-rium, which might either be due to adsorption of toxic substances produced byS. Typhimurium (Singh and Sharma 1999; Siddiqui et al. 2004) on seed or tomovement of the pathogen from seeds to soil, and needs further investigation.However, no effect of killed Salmonella on germination of cowpea seedsindicated that either lipopolysaccharide or other heat-resistant moieties wereresponsible for the phytopathic effect of Salmonella. On the third day ofsowing, germination of cowpea in all pots of nonsterile soil was significantlyless (P < 0.01) than that in pots of sterile soil. However, after 7 days of sowing,the difference was insignificant. The observations suggested that othermicrobes in soil might act in concert with S. Typhimurium to slow downgermination. Little is understood about the potential of Salmonella to affectother crops; recent studies have shown that Salmonella does not affect differ-ent plants similarly, and internalization of Salmonella was observed in lettuceand radish but not in cress or spinach seedlings (Jablasone et al. 2005). It isevident from the observations that S. Typhimurium has some phytopathogenicpotential, which is of utmost significant in view of its zoonotic potential.Studies to determine the phytopathogenic potential of different strains of S.Typhimurium and other zoonotic Salmonella on different crops may be an


important area of research and may be needed for understanding the epidemi-ology of salmonellosis.

Although S. Typhimurium persisted in plants grown from Salmonella-inoculated seeds as well as in Salmonella-inoculated soil throughout the obser-vation period, its population in plants grown from inoculated seeds in sterilesoil was significantly higher (P < 0.05), particularly in aerial parts, than inplants of other groups. This might be due to the flourishing of S. Typhimuriumin the lack of competition with resident soil organisms and inhibitorysubstances.

Gentamicin treatment (to kill epiphytic Salmonella) of cowpea plants ofA group resulted in considerable reduction of S. Typhimurium but not onplants of B, C or D groups. The results indicated that most of the bacterial cellswere inside the plant tissues (endophytic) when plants were grown either innonsterile soil or in Salmonella-contaminated soil. The observations are inconcurrence with earlier observations of endophytic presence of Salmonellain alfalfa plants grown in nonsterile soil (Yuemei et al. 2003).

Persistence of Salmonella in aerial parts of cowpea plants exposed to S.Typhimurium postgermination showed a declining trend with increasing ageof plants. In 21-day-old plants, S. Typhimurium could not be detected in aerialparts for more than 3 h of irrigation of plants with S. Typhimurium-contaminated water (Table 4). However, S. Typhimurium persisted throughoutthe study period in stumps of the cowpea plants irrespective of age at the timeof irrigation with S. Typhimurium-contaminated water. The results corroboratethe findings of earlier workers reporting the persistence of Salmonella up to a10-cm height on grasses growing in Salmonella-contaminated pastures (Raschand Richter 1956; Jeck and Hepper 1969).

Rapid elimination of S. Typhimurium from the aerial parts of cowpeaplants exposed to the pathogen after 5 days of sowing, but continuous survivalof the organism in basal parts may be due to development of some kind ofresistance in older plants. Resistance to pathogens in plants has been proposedto be due to development of protective coating on various channels (Samishet al. 1962) of the plants with advancing age, not allowing pathogens such asSalmonella to enter plant tissues. Those few Salmonella that entered the aerialparts of the plants exposed to Salmonella at 10–21 days of age might not havebeen maintained because of some injury or stress in traveling through thetough-coated channels; however, to conclude, a more detailed study isrequired. Observation of life-long persistence of S. Typhimurium in top leavesof plants grown from inoculated seeds or seeds sown in inoculated soil may bedue to repeated invasion of Salmonella in new growths, probably due tosusceptibility of growing parts of plants to invasion by Salmonella. The studysuggested that plants grown in a Salmonella-free state, although susceptiblefor invasion of Salmonella just after contamination of soil, did not allow


Salmonella to persist in their aerial parts, and the knowledge can be exploitedto grow Salmonella-free crops by transplanting saplings grown in Salmonella-free soil.

The findings clearly revealed the phytopathogenic potential of S. Typh-imurium for cowpea and long-term persistence of the pathogen in plants grownin a contaminated environment or from contaminated seeds. It was evident thateven after the death of a plant, Salmonella persists for a long period in planttissues as S. Typhimurium persisted for 60 days in contaminated cowpea hay.Persistence of Salmonella in hay may be hazardous to persons handling hayand to animals eating it. Thus, it can be concluded that the presence of largenumbers of Salmonella either on seeds or in farm soil (which is possiblebecause of contamination of soil after irrigation with a sewage or on opendefecation by the excretor in the vegetable field) may be of high public andanimal health significance as the pathogen can grow and persist in fodder andgreen vegetable crops too.

ACKNOWLEDGMENT

The funds provided by the director of the Indian Veterinary ResearchInstitute, Izatnagar, for the study, are thankfully acknowledged.

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