INTERACTIONS BETWEEN SALMONELLA ENTERICA SUBSPECIES ENTERICA SEROVAR TYPHIMURIUM AND COWPEA (VIGNA UNGUICULATA VARIETY SINENSIS) SEEDS, PLANTS AND PERSISTENCE IN HAY

INTERACTIONS BETWEEN SALMONELLA ENTERICA SUBSPECIES ENTERICA SEROVAR TYPHIMURIUM AND COWPEA (VIGNA UNGUICULATA VARIETY SINENSIS) SEEDS, PLANTS AND PERSISTENCE IN HAY BHOJ RAJ SINGH1, MUDIT CHANDRA, RAVIKANT AGARWAL and NAGRAJAN BABU National Salmonella Center (Veterinary) Division of Bacteriology and Mycology Indian Veterinary Research Institute Izatnagar-243 122, India Accepted for Publication September 18, 2006

ABSTRACT Dynamics of persistence of Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium), a common food- and waterborne zoonotic serovar, on cowpea (Vigna unguiculata variety sinensis), a fodder and green vegetable plant, were studied. The findings revealed that S. Typhimurium not only reduced germination of cowpea seed (P < 0.01), but also caused defects in sprouts (P < 0.02). S. Typhimurium inoculation on seeds before sowing had a more pronounced effect (P < 0.01; i.e., loss in germination and appearance of defective sprouts) than sowing seeds in S. Typhimurium-inoculated soil. S. Typhimurium persisted in saplings and adult plants up to 45 days of plant age and up to 60 days in hay. The cowpea plants grown in sterile Salmonella-free soil did not support colonization of S. Typhimurium in different parts. A reduction in the population of Salmonella appeared as early as on the fifth day and decreased with advancing plant age. At 21 days of age, the cowpea plants had no Salmonella in their 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 study revealed the persistence and the phytopathogenic potential of Salmonella on cowpea.

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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: 0091-1662276217. EMAIL: [email protected]

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

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PRACTICAL APPLICATIONS The results of the study will be useful in understanding the epidemiology of salmonellosis, particularly as a foodborne pathogen, and the revelation of the phytopathogenic potential of Salmonella for cowpea opens a new vista for researchers to understand the mechanism of pathogenesis of salmonellosis, knowledge of which may be applied for other plant pathogens. The results indicating that seedlings grown in Salmonella-free soil are resistant to invasion of Salmonella in their later life may be exploited for raising Salmonella-free plants or vegetable products and may have impressive economic impact on agro-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 millions of livestock become carriers of Salmonella to act as a source of the pathogen to the human population and other animals. There are many sources of infection to animals, including environment, feeds and fodder (Wray and Davies 2000). Isolation of Salmonella from different feed ingredients of vegetable origin is being increasingly reported (DEFRA 2004); consumption of contaminated feed results in either outbreaks of apparent disease or animals becoming carriers (Wray and Davies 2000; DEFRA 2004). Studies on pasture land grasses (Taylor and Burrow 1971; Kelly and Collins 1978), maize (Singh et al. 2004) and tomato plants (Guo et al. 2001, 2002) have shown that Salmonella travels through and persists in plant tissues, and that pasture contamination may be the cause of disease outbreaks (Rasch and Richter 1956; Jeck and Hepper 1969). From the contaminated soil, Salmonella has been shown to reach the susceptible hosts via contaminated grasses, vegetables and sprouts (Clegg et al. 1983). Seeds have been reported to be the major source of Salmonella in sprouts (NACMCF/FDA 1999). Enteric pathogens have been shown to reside within the interiors of apples, lettuce and maize plants, and alfalfa 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 Salmonella Typhimurium and other enteropathogens on alfalfa and Lucerne (Medicago sativa and Medicago truncatula) plants and maize plants (Singh et al. 2004) demonstrated the rhizosphereic and endophytic colonization of enteropathogens to a variable extent depending on enteropathogenic strains. Cowpea (Vigna unguiculata variety sinensis), a multiseasonal legume crop, is native to

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India and the Middle East. It is extensively grown as a hay crop and greenmanure crop, and the beans (used as green vegetable) for human consumption. However, little is known about the effect of Salmonella on cowpea seeds and plants. 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 of poultry birds, which died in a maize grain-associated outbreak, by growing the pathogen in trypticase soy broth (TSB, Becton Dickinson and Co., Sparks, NV) as described earlier (Fett and Cooke 2003). Bacterial cells were harvested by centrifugation (5,000 ¥ g, 15 min), and the cell pellet was suspended in 0.01 M sodium phosphate buffer (PBS, pH 7.2). Colony-forming units (cfu) of S. Typhimurium in the inoculum were determined by plating serially diluted (10-fold dilutions made in PBS) aliquots in triplicate on HEA (Hektoen enteric agar, Becton Dickinson and Co.) plates. Each fresh inoculum was prepared from 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 by heating 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 incubated 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-L capacity were filled with 3 kg of potting soil procured from the section of Horticulture, Indian Veterinary Research Institute, Izatnagar. To sterilize the soil, pots with soil were autoclaved at 121C for 30 min and then placed in a polypropylene tray (AK Scientific Industries). To inoculate the pot-soil with S. Typhimurium, the pots were irrigated with 500 mL of water containing 9.8 ¥ 107 cfu of S. Typhimurium/mL 7 days before sowing. The control pots were irrigated with 500 mL of water containing an equal amount of heat-killed Salmonella 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 from insects, pests and rainwater. Room air and soil were free of Salmonella, and it was affirmed at monthly intervals through multiple site soil testing and air sampling for Salmonella (Edwards and Ewing 1986; Senior 1996). No arrangements were made to control humidity or temperature because the experiment was carried out during the cowpea cropping season (June to September).

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Salmonella Count in Soil, Seed, Seedlings and Plants Plants or their parts were harvested aseptically in individual sterile containers using a separate set of sterilized forceps, spatula and scissors. Ten grams of seeds/seedlings/plant tissues were homogenized individually in 100 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 in homogenates were determined by plating 100 mL of serially diluted (in PBS) aliquots in triplicate on HEA plates (Becton Dickinson and Co.); suspected colonies were confirmed through plate agglutination test with specific (O factor 5 and H factor i and 2) serum (Edwards and Ewing 1986). Ten milliliter of homogenates was also inoculated into 90 mL of universal preenrichment broth (Becton Dickinson and Co.) and was incubated at 37C for 16 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 Vasilliadis medium (RV, Becton Dickinson and Co.), and was incubated at 37C for 24 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 for detecting Salmonella colonies. Suspected colonies were confirmed as mentioned earlier. For determining S. Typhimurium in soil, 10 g of soil was suspended by whirling in 100 mL of sterile distilled water, and then the suspension was processed similar to plant tissue homogenate. Five presumptive colonies were confirmed by agglutination with O factor 5 and H factor i and 2 specific serum (Becton Dickinson and Co.) according to the manufacturer’s instructions.

Evaluation of Effect of S. Typhimurium on Germination of Cowpea Certified seed of cowpea cultivar UPC-607, purchased from a retail outlet of GB Pant University of Agriculture and Technology, Pantnagar, Uttaranchal, were checked for the presence of Salmonella as described earlier by taking three samples of 25 g each. The seeds were randomly divided in groups of 100 seeds each. Treatments to seeds of different groups are summarized in Table 1. After various treatments, excess fluid was drained off and seeds were placed on six layers of wet cheesecloth placed on seed-germinating plates (AK Scientific Industries) for 48 h in the dark at 30C. To determine the rate of germination, the number of seeds showing a visible radicle (root) and opening to the hypocotyls was counted. Salmonella count was made before and after gentamicin treatment to determine the endophytic and epiphytic population of Salmonella in germinating seeds using the method of Singh et al. (2005).

Soaked in 100 mL of aqueous solution of gentamicin (200 mg/mL) for 30 min.

E Soaked in 100 mL of distilled water containing ~7.2 ¥ 106 cfu/mL S. Typhimurium for 30 min at 30C.

Soaked in 100 mL of distilled water for 30 min.

F Soaked in 100 mL of distilled water containing ~7.2 ¥ 106 cfu/mL S. Typhimurium for 30 min at 30C.

Salmonella counted in germinated as well as in nongerminated seeds.

Washed with sterilized distilled water then twice with PBS.

Washed with aqueous solution of gentamicin (200 mg/mL) for 30 min and then twice with PBS.

To determine the rate of germination, the number of seeds giving out a visible radicle (root) and opening of the hypocotyls was counted.

Seeds were placed on six layers of wet cheesecloth then placed on seed-germinating plates (AK Scientific Industries) for 48 h in the dark at 30C.

No treatment

Washed twice with sterilized distilled water and left at room temperature for 4 h.

Salmonella-free certified seed of cowpea cultivar UPC-607 divided into six groups A B C D Soaked in 100 mL of Soaked in 100 mL of Soaked in 100 mL of Soaked in 100 mL of distilled water distilled water distilled water distilled water containing containing containing containing heat-killed ~7.2 ¥ 104 cfu/mL ~7.2 ¥ 106 cfu/mL ~7.2 ¥ 106 cfu/mL (~7.2 ¥ 106 cfu/mL) S. Typhimurium for S. Typhimurium for S. Typhimurium for S. Typhimurium for 30 min at 30C. 30 min at 30C. 30 min at 30C. 30 min at 30C.

TABLE 1. EXPERIMENTAL PLAN FOR EVALUATION OF EFFECT OF SALMONELLA TYPHIMURIUM ON GERMINATION OF COWPEA SEEDS INTERACTION OF SALMONELLA WITH COWPEA 173

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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 water containing 7.2 ¥ 106 cfu of S. Typhimurium/mL for 30 min and then washed twice with sterilized distilled water as mentioned earlier. In each pot, 15 seeds were sown. Thirty-six pots were divided into groups A to F (Table 2); six pots in each group had either sterile (A, C, E) or nonsterile soil (B, D, F) and were either inoculated with Salmonella (C, D) or not inoculated with Salmonella (A, B, E, F). Observations were made for number of seeds germinated (on the third, 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 above base, 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 and 45th day of sowing) were harvested aseptically as mentioned earlier. To count the endophytic S. Typhimurium, three plants from each group were cut at a height of 5 cm from the ground and dipped into 1,000 mL of aqueous solution of gentamicin (200 mg/mL) for 30 min, leaving the cut surface outside the solution (Singh et al. 2005). Thereafter, the plants were taken out and the undipped portion was clipped off. The remainder of the plant was washed twice with sterilized distilled water (1,000 mL) and was processed for counting Salmonella. Similarly, the plants from the respective pots, but not treated with gentamicin, were used as controls. Evaluation of Effect of Age on Susceptibility of Cowpea Plants to S. Typhimurium Inoculation To determine the number of Salmonella invading and surviving in different parts of the cowpea plants at different stages of growth, each of the 24 pots with sterile soil was seeded with 15 Salmonella-free cowpea seeds and divided equally 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 were used for irrigation of groups 1, 2, 3, 4 and 5 pots on the 0, 5th, 10th, 15th and 21st days of sowing, respectively. For irrigation, 500 mL of sterile distilled water was used after 2 h of sowing and thereafter at a weekly interval. For Salmonella counts in plants, three plants from each of the group were cut at a height of 5 cm from the base. On the day of S. Typhimurium inoculation, the plants were cut at 1, 2, 3, 6, 12, 18 and 24 h and divided into three parts, namely the basal (10 cm of stem), top leaves and branches with the rest of the stem. On the 5th, 10th and 21st days of Salmonella inoculation, the plants were cut and processed as whole for Salmonella count. On the 21st day of Salmonella inoculation, stumps of the plants were also pulled out and processed for Salmonella count determined after homogenization of plant tissues as described earlier.

Salmonella-free seeds

Observations were made for number of seeds germinated (on the third, fifth and seventh day of sowing), height of saplings (on the fifth day) and count of S. Typhimurium in different parts (leaves, stem cut 5 cm above base, 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 and 45th day of sowing) were harvested aseptically as mentioned earlier.

Salmonella-inoculated seeds

Each pot was seeded either with 15 Salmonella-free cowpea (cultivar UPC-607) seeds or with seeds inoculated with S. Typhimurium by soaking in water containing 7.2 ¥ 106 cfu of S. Typhimurium/mL for 30 min and then washed twice with sterilized distilled water.

Thirty-six soil pots were divided into six groups, pots contained 3 kg of either sterilized (autoclaved at 121C for 30 min) or nonsterilized Salmonella-free potting soil either inoculated or not inoculated with Salmonella enterica serovar Typhimurium (through irrigation with 500 mL of water containing 9.8 ¥ 107 cfu of S. Typhimurium/mL 7 days before sowing). A B C D E F Sterile Salmonella-free Nonsterile Sterile soil containing S. Nonsterile soil containing Sterile Salmonella-free Nonsterile soil Salmonella-free soil Typhimurium S. Typhimurium soil Salmonella-free soil

TABLE 2. EXPERIMENTAL PLANT FOR EVALUATION OF EFFECT OF SALMONELLA TYPHIMURIUM ON COWPEA SEEDS SOWN IN SOIL

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Survival of Salmonella on Hay To determine the survival of S. Typhimurium on hay of cowpea, 30 pots with sterile potting soil were sown with Salmonella-free cowpea seeds (five in each). 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 sterile distilled water 2 h after sowing. Thereafter, the sterile distilled water was used for all irrigations. After 45 days of growth, plants were harvested and placed in separate sterilized trays for 7 days to make hay in the greenhouse itself. Group A hay (moisture content 98% of S. Typhimurium got killed. Observations on Salmonella invasion in plant tissues revealed that inoculation of soil with Salmonella on the day of sowing and on the fifth day of sowing resulted in dissemination of Salmonella to all parts of the plants. The organism could be isolated even after 21 days of observation. In plants of the pots of groups 3, 4 and 5 inoculated with S. Typhimurium after 10, 15 and 21 days of sowing, the pathogen could be detected up to 12, 6 and 3 h, respectively after irrigation with Salmonella-contaminated water, and the pathogen could not be recovered from aerial parts of plants after 15 h (Table 4). S. Typhimurium persisted in cowpea plants in roots and stumps, irrespective of the 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 four groups (2–5) within an hour of irrigation with the Salmonella-contaminated water. On hay, S. Typhimurium persisted throughout the observation period of 60 days, irrespective of its mode of entry onto cowpea plants (Fig. 3). Hay from the plants grown in Salmonella-contaminated soil had 4.18 log10 cfu of S. Typhimurium/g. On the 10th day of haymaking, S. Typhimurium population was significantly (P < 0.01) less on hay made from plants grown in S. Typhimurium-contaminated soil than hay contaminated with Salmonella after harvest; however, from the 20th day onward, the difference in population of the 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

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TABLE 4. EFFECT OF AGE OF COWPEA (VIGNA UNGUICULATA VARIETY SINENSIS) PLANTS ON THEIR SUSCEPTIBILITY TO SALMONELLA TYPHIMURIUM Time after irrigation

1h

3h

6h

12 h

15 h

18 h

24 h

5 days 21 days

Parts of the plant

Top leaves Stem† Branches‡ Top leaves Stem† Branches‡ Top leaves Stem† Branches‡ Top leaves Stem† Branches‡ Top leaves Stem† Branches‡ Top leaves Stem† Branches‡ Top leaves Stem† Branches‡ Whole plant§ Whole plant¶ Whole plant§ Whole plant¶ Stumps** Stumps**

Salmonella count (cfu/g in log10) in plants irrigated with 500 mL Salmonella (7.2 ¥ 106 cfu/mL) containing water at the age of 0 day*

5 days

10 days

15 days

21 days

5.56 (0.05) 4.51 (0.17) 4.54 (0.04) 4.42 (0.07) 3.83 (0.22) 3.89 (0.04) 3.77 (0.10) 2.74 (0.25) 3.13 (0.14) 3.62 (0.12) 2.40 (0.39) 2.83 (0.13) 3.39 (0.14) 2.13 (0.07) 2.09 (0.12) 3.11(0.04) AE (NC) AE (NC) 2.90 (0.06) AE (NC) AE (NC) 2.13 (0.28) 2.80 (0.15) 2.57 (0.38) 3.69 (0.20) 4.49 (0.11) 4.52 (0.08)

3.19 3.70 3.54 3.22 3.16 3.21 3.03 2.05 2.24 2.91 1.06 1.47 2.82 AE AE 2.27 AE AE AE AE AE AE AE 0.0 AE 3.88 4.26

AE 3.36 2.12 AE 3.13 2.80 2.35 AE 2.09 AE 0.0 AE 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.64 3.74

AE 3.39 3.36 AE 3.14 AE 0.0 AE 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.83 3.69

0.0 AE AE 0.0 AE 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.48 3.35

(0.19) (0.17) (0.09) (0.07) (0.08) (0.05) (0.01) (0.8) (0.09) (0.07) (1.24) (0.98) (0.09) (NC) (NC) (0.13) (NC) (NC) (NC) (NC) (NC) (NC) (NC) (0.00) (NC) (0.06) (0.19)

(NC) (0.22) (0.08) (NC) (0.07) (0.09) (0.27) (NC) (0.28) (NC) (0.00) (NC) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.23) (0.16)

(NC) (0.11) (0.10) (NC) (0.20) (NC) (0.00) (NC) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.09) (0.18)

(0.00) (NC) (NC) (0.00) (NC) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.04) (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 ⱖ1 cfu/g). NC, SDs were not calculated.

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

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Log10 of average CFUs gm of hay

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6

A

B

5 4 3 2 1 0 0

10

20

30

40

50

60

Days after haymaking FIG. 3. SURVIVAL OF SALMONELLA ENTERICA SUBSPECIES ENTERICA SEROVAR TYPHIMURIUM 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.

(common in most of the developing counties) may lead to heavy contamination of the soil, particularly at the defecation spot, and plants growing on that particular spot may be heavily infected with the pathogen. Sewage (Clegg et al. 1983) and composts (Smith et al. 1982) have been reported to be the sources of Salmonella and other zoonotic pathogens. It is often believed that animal and human pathogens persist on plants without harming them. Earlier studies suggested that Salmonella might enter into plants through abrasion on bark tissues, contamination of flowers and at early stages of development of fruits (Guo et al. 2002). Multiplication of Salmonella in different parts of alfalfa plants (Yuemei et al. 2003) has been reported recently. However, little is known about the fate of Salmonella on cowpea, a common leguminous green fodder and hay crop. Observation revealed that only live S. Typhimurium reduced the germination of cowpea seeds (P < 0.01) above a certain threshold level, which is in contrast to earlier studies reporting no effect of Salmonella on germination of alfalfa seeds (Fett and Cooke 2003). It may be either a result of the difference in phytopathogenic potential of S. Typhimurium strain or the susceptibility of 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 other Salmonella strains which may reduce germination of other cultivars of cowpea and probably of other crops too, and needs further studies. Gentamicin treatment of seeds after 4 h of inoculation with S. Typhimurium reduced the population of the pathogen by about 4 to 5 log10, indicating that the majority of Salmonella remained on the seed surface in the first 4 h of

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inoculation. After gentamicin treatment, the remaining 1.18 log10 cfu of Salmonella/g of seed multiplied to the extent of 7.6 log10 cfu of Salmonella/g on germinated and 9.16 log10 cfu of Salmonella/g on nongerminated seeds. Thus, the observations reflect that on invading into seeds, S. Typhimurium grew inside tissues of germinating seed and baby plants. These observations are in concurrence with earlier reports on multiplication of Escherichia coli and 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. Typhimurium after gentamicin treatment significantly (P < 0.01) improved the germination rate of S. Typhimurium-inoculated seeds. The observation revealed that not only endophytic (those that invaded the seed) but also epiphytic Salmonella have a role in inhibition of germination. It appears that S. Typhimurium cells (1.18 log10 cfu/g) that survived gentamicin treatment might have internalized in cowpea seeds and grew to sufficient numbers (9.2 log10 cfu/g) to inhibit the germination. Similar observations of internalization of S. Typhimurium in lettuce and radish seedlings grown from contaminated seeds have been reported recently (Jablasone et al. 2005). Nongerminating cowpea seeds had significantly (P < 0.05) higher numbers of S. Typhimurium than germinated sprouts, particularly in the gentamicin-treated group (Fig. 2), indicating a definite role of high load of Salmonella or its product in inhibiting the germination of cowpea seeds. Comparison between germination of cowpea seeds sown in soil and in sprouting plates revealed that soil gave some protection against S. Typhimurium, which might either be due to adsorption of toxic substances produced by S. Typhimurium (Singh and Sharma 1999; Siddiqui et al. 2004) on seed or to movement of the pathogen from seeds to soil, and needs further investigation. However, no effect of killed Salmonella on germination of cowpea seeds indicated that either lipopolysaccharide or other heat-resistant moieties were responsible for the phytopathic effect of Salmonella. On the third day of sowing, germination of cowpea in all pots of nonsterile soil was significantly less (P < 0.01) than that in pots of sterile soil. However, after 7 days of sowing, the difference was insignificant. The observations suggested that other microbes in soil might act in concert with S. Typhimurium to slow down germination. Little is understood about the potential of Salmonella to affect other crops; recent studies have shown that Salmonella does not affect different plants similarly, and internalization of Salmonella was observed in lettuce and radish but not in cress or spinach seedlings (Jablasone et al. 2005). It is evident from the observations that S. Typhimurium has some phytopathogenic potential, 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

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important area of research and may be needed for understanding the epidemiology of salmonellosis. Although S. Typhimurium persisted in plants grown from Salmonellainoculated seeds as well as in Salmonella-inoculated soil throughout the observation period, its population in plants grown from inoculated seeds in sterile soil was significantly higher (P < 0.05), particularly in aerial parts, than in plants of other groups. This might be due to the flourishing of S. Typhimurium in the lack of competition with resident soil organisms and inhibitory substances. Gentamicin treatment (to kill epiphytic Salmonella) of cowpea plants of A group resulted in considerable reduction of S. Typhimurium but not on plants of B, C or D groups. The results indicated that most of the bacterial cells were inside the plant tissues (endophytic) when plants were grown either in nonsterile soil or in Salmonella-contaminated soil. The observations are in concurrence with earlier observations of endophytic presence of Salmonella in 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 age of plants. In 21-day-old plants, S. Typhimurium could not be detected in aerial parts for more than 3 h of irrigation of plants with S. Typhimuriumcontaminated water (Table 4). However, S. Typhimurium persisted throughout the study period in stumps of the cowpea plants irrespective of age at the time of irrigation with S. Typhimurium-contaminated water. The results corroborate the findings of earlier workers reporting the persistence of Salmonella up to a 10-cm height on grasses growing in Salmonella-contaminated pastures (Rasch and Richter 1956; Jeck and Hepper 1969). Rapid elimination of S. Typhimurium from the aerial parts of cowpea plants exposed to the pathogen after 5 days of sowing, but continuous survival of the organism in basal parts may be due to development of some kind of resistance in older plants. Resistance to pathogens in plants has been proposed to be due to development of protective coating on various channels (Samish et al. 1962) of the plants with advancing age, not allowing pathogens such as Salmonella to enter plant tissues. Those few Salmonella that entered the aerial parts of the plants exposed to Salmonella at 10–21 days of age might not have been maintained because of some injury or stress in traveling through the tough-coated channels; however, to conclude, a more detailed study is required. Observation of life-long persistence of S. Typhimurium in top leaves of plants grown from inoculated seeds or seeds sown in inoculated soil may be due to repeated invasion of Salmonella in new growths, probably due to susceptibility of growing parts of plants to invasion by Salmonella. The study suggested that plants grown in a Salmonella-free state, although susceptible for invasion of Salmonella just after contamination of soil, did not allow

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Salmonella to persist in their aerial parts, and the knowledge can be exploited to grow Salmonella-free crops by transplanting saplings grown in Salmonellafree soil. The findings clearly revealed the phytopathogenic potential of S. Typhimurium for cowpea and long-term persistence of the pathogen in plants grown in a contaminated environment or from contaminated seeds. It was evident that even after the death of a plant, Salmonella persists for a long period in plant tissues as S. Typhimurium persisted for 60 days in contaminated cowpea hay. Persistence of Salmonella in hay may be hazardous to persons handling hay and to animals eating it. Thus, it can be concluded that the presence of large numbers of Salmonella either on seeds or in farm soil (which is possible because of contamination of soil after irrigation with a sewage or on open defecation by the excretor in the vegetable field) may be of high public and animal health significance as the pathogen can grow and persist in fodder and green vegetable crops too.

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

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