Pathogenic effects of Salmonella enterica subspecies enterica serovar Typhimurium on sprouting and growth of maize

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1 Indian Journal of Experimental Biology Vol. 42, November 24, pp Pathogenic effects of Salmonella enterica subspecies enterica serovar Typhimurium on sprouting and growth of maize B R Singh*, Ravikant Agarwal & Mudit Chandra National Salmonella Centre (Vet.), Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar , India Received 23 December 23; revised 5 August 24 The study was undertaken to understand effects and survival of S. enterica subspecies enterica serovar Typhimurium (S. Typhimurium), a zoonotic serovar, on maize seed germination and plant growth. All the four strains of S. enterica subspecies enterica serovar Typhimurium significantly reduced germination of maize seeds in sprouting plates as well as in soil. About 2:2.7xl 3 Salmonella" cfu ml" 1 of soaking water, while 2:2.7x 1 7 Salmonella cfu g 1 soil were required to significantly inhibit germination of maize. Similar inhibition of germination could be observed using 2: 16 mg of bacteria free Salmonella cell lysate (CL) protein per g of soil or 2:.5 mg of CL protein per ml of soaking water in sprouting plates. At the constant dose of 3.6xl 7 to 3,8x1 7 Salmonella cfu or 5 mg cell lysate protein ml" 1 of soaking water, four strains of Salmonella significantly reduced germination, however difference between strains was insignificant. After germination too, mai ze growth was affected both by Salmonella organism and CL with little strain-to-strain variation. All Salmonella persisted in growing plants from 15 to 35 days of plant age and up to 19 days in soil. Maize plants once grown for a week in sterile soil were resistant to invasion of S. enterica subspecies enterica serovar Typhimurium in their leaves even in doses as high as 7.6xl 9 cfu g 1 of soil. Salmonella persisted better and longer in plants grown from contaminated seed sown in loam soil, but rarely in plants grew in sandy soil. All maize plants had Salmonella in their stumps even after 35 days of sowing irrespective of kind of soil, primary source of infection (soil or seed) and type of S. e!uerica subspecies enterica serovar Typhimurium strain. The study revealed that Salmonella is not only zoonotic but a phytopathogen also. Keywords: Salmonella, Phyto-pathogenicity, Germination, Persistence of Salmonella Every year Salmonella infect millions of livestock of which thousands of animals suffer from the disease and many become carriers of Salmonella. There are many different sources of infection for animals viz., environment, feeds and fodder 1 Isolation of Salmonella, more often S. enterica subspecies enterica serovar Typhimurium (S. Typhimurium), from different feed ingredients of vegetable origin is common. Many a time consumption of contaminated feed results either in outbreaks of apparent disease or animals become carriers 2 3. How Salmonella get entry into vegetable products is still a puzzle? However, studies on pasture land grasses 4 ' 5 and tomato plants 6 7 have shown that Salmonella persists and migrates in plant tissues and may be the cause of disease outbreaks 8 9. There is only scanty knowledge about pathogenic effects of Salmonella on fodder plants. *Correspondent author: Phone: (), (R) Fax: singh_br 1762@rediffmail.com; brsl762@ ivri.up.nic.in Earlier studies have suggested that Salmonella can enter in fruits and probably other parts of plants through abrasions in plant tissues and contamination of flowers at early stages of development of fruits 7, but nothing is known about the fate of Salmonella on fodder crops and fate of fodder crops in Salmonella contaminated lands. This study has been undertaken to evaluate the ill effects of Salmonella Typhimurium on maize, a multi-seasonal fast growing fodder crop commonly grown in India and elsewhere. The study has evaluated effect of contamination of seeds and soil (at different stages of cropping) on the germination and growth of maize. Survival of Salmonella in different parts of plants was also studied. Materials and Methods Bacterial culture and preparation of inoculums--inocuiums were made by growing frozen stock of Salmonella enterica serotype Typhimurium (E2391, E2597, E1116, isolated from food/ feeds and E42, a reference pathogenic strain) in trypticase soy

2 SINGH et.al: PHYTO-PATHOGENICITY OF SALMONELLA TYPHIMURIUM 111 broth (Difco) for 24 hr at 37 C 1. The broth culture was pelleted by centrifugation at 5 g and washed twice with 15 ml of.1 M sodium phosphate buffer (ph 7.2) containing.85% sodium chloride (PBS, phosphate-buffered saline) and suspension of bacterial cells was made in PBS to the OD 5 4 of.6. Colony forming units (cfu) of Salmonella in inoculums were determined by plating serially diluted aliquots in triplicate on HEA (Hektoen enteric agar, Difco, BBL) plates. Every time a fresh culture from single stock was prepared for inoculation of soil, water or seeds. Preparation of pots-autoclaved plastic pots (A K Scientific Industries, Delhi, India) of capacity were filled with 75 g loam soil with 3% humus or sandy (particle size I to diam.) soil (Section of Horticulture, Indian Veterinary Research Institute, Izatnagar). Pots were autoclaved at 121 C for 3 min and then placed in a polypropylene tray (A K Scientific Industries, Delhi, India) to retain water that filtered through the soil after application of water to the soil surface. To contaminate pot soil with Salmonella or its cell lysate pots were irrigated with 1 ml of water contaminated with Salmonella or Salmonella cell lysate 11 respectively. Contaminated pots were made 7 days before sowing. Other pots for use were irrigated with equal amount of sterilized water. Pots were contaminated with required strain at about 3.6x1 7 cfu g 1 of soil. For post sowing contamination, Salmonella contaminated water was applied as above. Post sowing contamination of pots was done either 2 hr post sowing or 1 days post sowing. All pots and plants were kept in an airy room with fiber glass roof to protect from insects, pests, and rain water, no arrangements were made to control humidity or temperature because the experiment was done in natural maize cropping season in the month of June to September for two consecutive years. Salmonella count in soil and plants-about 1 g or available amount (weighed) of plant tissues were homogenized in 1 volumes of PBS with Ultra Turrax T-25 (IKA, Werke Gmbh & Co. KG, Germany) homogenizer for 1 min. Colony forming units (cfu) of Salmonella in homogenates were determined by plating serially diluted aliquots in triplicate on HEA plates. Plates were incubated at 37 C for 24 hr before presumptive colony counting. Besides counting, homogenates were also enriched for Salmonella in universal pre-enrichment broth (Becton Dickinson and Co. Sparks, USA) at 37 C for 24 hr, then one ml aliquot was transferred to TTB (Becton Dickinson and Co. Sparks, USA) and incubated at 37 C for 24 hr. TTB cultures were streaked on HEA plates and incubated at 37 C for 24 hr for counting presumptive Salmonella colonies. For Salmonella count in soil, 1 g of soil was suspended by whirling it in 9 ml of sterile water and then processed further like plant tissue homogenate described above. Five presumptive colonies were randomly picked from each plate and transferred to TSA. Serological identification was performed by using Salmonella antiserum against factor '5' and H factor 'i' or '2' (Becton Dickinson and Co. Sparks, USA) according to the manufacturer's instructions. Evaluation of effect of Salmonella on germination/ sprouting--certified Maize variety 'Ganga' seeds purchased from retail outlet of GB Pant University of Agriculture and Technology, Pantnagar, Uttranchal and stored at room temperature (32 ± 7.2 C) in dry place were distributed in groups of 1 seeds. For determination of approximate contamination level of S. Typhimurium (E-2391) required to inhibit germination in sprouting plates, groups of seeds were soaked in 1 ml of water contaminated with 1-fold diluted (to contain 2.7 to 2.7x1 7 cfu of Salmonella mr 1 ) E-2391 culture in sterile water and control group seeds were soaked in 1 ml of sterile water for 4 hr at 3 C and then washed twice with sterile water and kept on wet sterilized muslin cloth laid in sprouting plates (A K Scientific Industries, Delhi, India) for sprouting of seed for 72 hr and sown in sterile soil pots for germination and observed after 4 days, seeds giving out visible radicle (root) and shoots after putting sprouting plates/soil pots at 3 C. Similarly, effect of cell lysate was assessed by soaking seeds in serially diluted (two fold) cell lysate made 11 from E2391 strain containing.125 mg to 16 mg of CL protein in each ml of preparation. To assess the effect of in-soil exposure of maize seed to E-2391 or its CL, soil (loam soil with 3% humus) was contaminated with Salmonella (to contain 2.7 to 2.7x1 7 cfu of Salmonella g' 1 ) or CL (to hold.125 mg to 16 mg of CL protein g' 1 ) through irrigation of soil with contaminated water to achieve same level of contamination as in water used for contaminating seed to be sown in sprouting plates and maize seeds were sown without soaking. After determining the effective dose of Salmonella (E-2391) and its CL, 3 more strains were compared for their effect on maize germination by presoaking maize seed in water contaminated with xl 7

3 112 INDIAN J EXP BIOL, NOVEMBER 24 cfu of Salmonella or 5 mg of CL protein ml" 1 of soaking water. Seeds were sown as described earlier in soil for observing germination after 4 days and growth of shoots was measured after 5, 1 and 15 days of sowing. Evaluation of effect of Salmonella contamination of soil/seeds on maize plants-the experiment was set in three sets, one with Salmonella soaked seeds (as described above), 2"d with contaminated soil after 2 hr of sowing and 3'd group pots were contaminated with Salmonella after 1 days of sowing. For each of the four strain two types of soil, loam soil with 3% humus and sandy soil (crystal size 1-3 J.l diam.) without humus were used. For both types of soil noncontaminated pots were used as control, each group had 4 pots each sown with 1 seeds. Observations were made for seeds germination at 4th day, presence of Salmonella in shoots (harvested above 1 em from ground) at 15th and 21st day, in stumps at 35 1 h day and in soil at 35th and 19th day of sowing as described above. Statistical analysis-the mean values and standard deviations for seed germination, defective sprouts, Salmonella counts in different parts of maize plants and at different times for various treatments were calculated using results of replicates. The results from various treatments were compared with control and with each other using paired two tailed Student's t test at 1 and 5% level of significance using Microsoft Exel-2. Results and Discussion The maize plants were adversely affected by Salmonella during early plant life (Fig. la). Contamination of maize seeds with Salmonella enterica serovar Typhimurium (S. Typhimurium) strain E-2391 containing water significantly (P<.1) lowered germination during sprouting over three days of observation in sprouting plates. The effect was dose dependent (Fig.l); and significant (P<.5 or less) effect was evident with minimum infective dose of 2:2.7x1 3 cfu of S. Typhimurium per ml of soaking water. Earlier studies on alfalfa 1 ' 13 revealed no difference in germination of seed before and after treatment (to remove Salmonella) of Salmonella inoculated seed, probably the detrimental effect of Salmonella as well as of treatments to remove Salmonella were equally detrimental for the germination of seed 12. However, significant reduction has been reported in germination of alfalfa seeds after Salmonella and E. coli inoculation 1 ' 13 which also supports the observations of the present study. Like S. Typhimurium bacteria, its CL also affected germination (Fig. lb) significantly at concentration 2:.5 mg CL protein mr 1 of seed soaking water. The observations indicated that some of the product(s) of Salmonella was responsible for inhibition of growth of sprout probably similar to Klebsiella cytotoxins inhibiting germination of maize seed 14 Salmonella cytotoxins are similar to Klebsiella cytotoxins in their cytotoxic effects on Vero and MDBK cells but are antigenically unrelated 11 ' 15 ' 16 Cell lysates of all h. S l ll h II pat ogemc a mone a are nc m cytotoxms however, to prove that the cytotoxins of Salmonella are the cause of inhibition of germination require further studies. Though CL and Salmonella significantly reduced germination of maize seed in sprouting plates at very low doses, a high concentration of Salmonella (2:2.7x1 7 cfu g r') or CL protein (2:16 mg g- 1 ) was required in soil to significantly lower the germination rate of maize (Figs la and lb). The exact cause behind requirement of high concentration of Salmonella or CL in soil to inhibit germination may be known by further studies; probably adsorption of toxic products of Salmonella to soil may be the putative reason. Further studies on growth and germination of maize seed in soil after contaminating seed with different strains (3.6x1 7 to 3.9x1 7 mr' of soaking water) of S. Typhimurium or with their CL preparations (5 mg of CL protein mr' of soaking water) revealed that maize seed germination was lowered significantly (P<.1) by all strains and their CL preparations (Fig. 2A), however, strain to strain variation was not significant. The study revealed that all Salmonella are potentially detrimental to seed as they are for human and animals 2. Although, all Salmonella and their CL preparations affected germination of seed to almost to the same extent, their effect on growth appeared to vary from strain to strain and there was significant difference (P<.5) in effect induced by CL preparatiobs of strains E2391 and E2597 and between CL of E42 and Ell16 at day 5 of sowing and later on also. Cell lysates of E2597 and E42 significantly (P<.5) depressed the growth of maize nearly to an equal extent (Fig. 2B). Such a marked strain variation was not evident when seeds were contaminated with Salmonella organism rather than CL which may be due to variation in concentration of phytotoxin in CL

4 SINGH et.al: PHYTO-PATHOGENICITY OF SALMONELLA TYPHIMURIUM 113 of different strains but all the strains may have released it to an equal level due to variability in extracellular release mechanism as reported earlier Studies in loam and sandy soil (Table 1) on precontaminated seeds (soaked m Salmonella contaminated water) sown in sterile soil, Salmonella free seeds sown in Salmonella contaminated soil and contamination of soil after 1 days of sowing revealed that Salmonella significantly inhibited germination of maize seeds irrespective of soil type when seeds were pre-contaminated but not when soil was contaminated except when the soil was contaminated after 2 hrs of sowing. Strain to strain variation was insignificant for inhibition of germination. Inhibition of germination was significantly (P<.1) high in sandy soil (Table 1). Protective effect of loam soil on seed germination would be attributed due to organic components present in the loam soil or the clay particle present in loam soil may have adsorbed the toxic moiety of Salmonella, but nothing can be concluded without further studies. All the strains of Salmonella persisted in plants grown from precontaminated seed in loam soil for 15 or more days. However, plants grown in sandy soil rarely yielded Salmonella from their aerial parts, except those which grew from seed contaminated with Salmonella strain E Salmonella counts in plants varied significantly (P<.5) from strain to strain and maximum numbers were detected for strain E-42. Till 1 em height i.e. in stumps of the plants, all Salmonella strains persisted even on 35 days of observation. In soil also all the strains persisted up to e-- In soil exposure " Q) Q) II) Q) N n; E In sprouting plate exposure 1 o , ~ ---r~._~-- ~~~ ~~ i Log 1 of cfu of S. Typhimurium I ml of soaking water or/ g of soil ~In soil exposure ~ - In sprouting plate exposure Concentration of CL protein (in mg ) I ml of soaking water or/ g of soil Fig. 1-Effect of lives. Typhimurium E2391 strain (A) and its cell lysate (B) on gennination of maize seed sown in sprouting plates and soil.

5 114 INDIAN J EXP BIOL, NOVEMBER days of observation except for sandy soil sown with contaminated seeds. In sandy soil, Salmonella probably did not move from seed to the soil for want of nutrients, however, Salmonella persisted in sandy soil contaminated with Salmonella in significantly (P<.1) low numbers for same length of time as in the loam soil. Persistence of Salmonella in soil and grasses growing on contaminated pasture lands has been reported 8 ' 9 The present study has also shown that Salmonella can persist in fodder plants on cultivated land. They have the ability to survive on grasses as well as on fodder crops to reach their final host. The earlier studies 8 ' 9 reported that Salmonella persist in grass up to the height of 1 em only, however we found Salmonella (P<.1) in top leaves also. The difference in observations is possibly due to the difference in mode of propagation of grass (mostly vegetative through suckers) and fodder crops (mostly through seeds). This was supported by the observations on post germination exposure of plants to Salmonella (Table 1). Age of the plants appeared to be the most crucial factor in determining the fate of Salmonella. As the age advanced, maize plants became resistant for permitting Salmonella to survive in their shoot system above 1 em of height. The observation of Salmonella persistence in stumps corroborates earlier findings 8 9 of persistence of Salmonella in grasses up to 1 em of height. Salmonella persisted in stumps of all the plants whether the seed or the soil was contaminated indicating that Salmonella was taken up by the maize plants from the soil probably through the root system and the pathogen is capable of migrating upward to some extent. Survival of Salmonella in basal parts of the maize plants and probably in other plants also 8 9 may be attributed to some kind of Salmonella killing/ 1., eo :: 7 ;;; E 6 5 ~ c 9 4 ~ c '[! 3 (!) Nobacleria B-E2391 B-C2597 B-E42 BE 1116 CL-C2391 CL-C2597 CL-E42 CL-E 1116 S. enfltrica SlbspeQes enterica serovartyptill'ljiiunstrairs 7.5 I 7 :E c -; 6 ~ ~ _, ~ --&-No bacteria - B-E B-E2597 -* -B-E42 - -BE ~ - CL-E2391 -cl-e2597 -<>-CL-E42 -X-CL-E T day 5 day 1 Log 2 of average shoot length ( mm ) Fig. 2--Effect of different S. Typhimurium strains and their cell lysates on germination (A) and growth (B) of maize seed sown in sprouting plates and soil, respectively.

6 SINGH et.al: PHYTO-PATHOGENICITY OF SALMONELLA TYPillMURIUM 115 Tablel-Effect of different S. Typhimurium strains on germination of seed and persistence of the pathogen in maize plant and soil Salmonella Soil Exp. Average% l5'h day day- 35'h day- 35'h day- l9'h daystrain type time gennination Salmonella Salmonella Salmonella Salmonella Salmonella in plants in plants in stumps in soil in soil E239l Sandy PS 5. (5.77) (.) (.) 4.51 (.2) 2.. (.) AS 5. (5.77) (.) (.) 4.53 (.3) 5.7 (.1) 3.67 (.14) Loam PS.5 (.58) 2.99 (.4) 2. (.) 4.56 (.4) 5.9 (.13) 4.81 (.4) AS 7. (8.16). (.). (.) 4.45 (.5) 5.37 (.16) 4.35 (.6) AG 98.5 (5.77). (.). (.) 5.47 (.6) 7.27 (.13) 6.32 (.17) E2597 Sandy PS 5. (5.) 1.91 (.53). (.) 4.49 (.6).67 (1.15). (.) AS 2.5 (5.77) (.) (.) 4.49 (.3) 4.4 (.13) 4.31 (.2) Loam PS 15. (5.77) 2. (.). (.) 4.54 (.2) 3.91 (.13) 4.73 (.6) AS 65. (5.). (.). (.) 4.15 (.4) 5.3 (.7) 4.54 (.9) AG 97. (5.77). (.). (.) 5.35 (.6) 7.42 (.53) 5.43 (.1) E42 Sandy PS 2.5 (5.77) (.) (.) 4.8 (.12). (.). (.) AS 5. (5.) (.) (.) 4.3 (.7) 2.18 (.9) 2.23 (.13) Loam PS 15. (5.) 3.35 (.31) 4.3 (.31) 4.78 (.13) 5.77 (.4) 5.59 (.1) AS 67.5 (9.77) 2. (.). (.) 4.46 (.11) 5.92 (.6) 5.59 (.3) AG 98. (9.77). (.). (.) 5.56 (.2) 7.41 (.9) 6.42 (.23) E1116 Sandy PS 2.5 (5.) (.) (.) 4.64 (.7).. (.) AS 2.5 (5.) (.) (.) 4.3 (.13) 2.34 (.9) 2.22 (.13) Loam PS 17.5 (5.) 2. (.). (.) 4.38 (.12) 5.5 (.4) 4.78 (.11) AS 7. (5.). (.). (.) 4.52 (.9) 7.24 (.25) 5.35 (.14) AG 98. (9.77). (.). (.) 5.32 (.16) 7.53 (.33) 6.43 (.17) No Sandy 92.5 (5.). (.). (.). (.). (.). (.) Salmonella Loam 97.5 (5.). (.). (.). (.). (.). (.) PS= pre-sowing soaking of seed in Salmonella contaminated water, AS= soil pots were irrigated with contaminated water after 2 hr of sowing, AG= soil pots were irrigated with contaminated water after 1 week of germination of maize seed i.e. after 1 days of sowing. No effect of Salmonella was evident on plant growth when soil was contaminated (7.6xl 9 cfu g- 1 of soil) after one week of germination of maize plant (1 days after sowing) and Salmonella was present only in stumps. eliminating activity in aerial parts which is absent in basal parts. Why resistance occurred in aerial parts needs more investigations. Resistance could not be attributed completely to development of protective coating on various channels 18 of the plants with advancing age, not allowing Salmonella to enter plant tissues because Salmonella persisted in plants which were containing Salmonella at the time of germination. The present study indicated that Salmonella Typhimurium decreases germination rate of maize seeds and growth of maize plants and Salmonella Typhimurium persists throughout plant life in basal part of the stem i.e. stumps. Thus, presence of Salmonella either on seeds or in soil for cultivation is of high public and animal health significance. Acknowledgement Thanks are due to the Director, IVRI, lzatnagar for financial assistance. References I Wray C & Davies R H, Salmonella infection in cattle, in Salmonellosis in domestic animals, edited by C Wray and A Wray ( CABI Publishing, Wallingford, Oxon) 2, Wray C & Wray A, Salmonellosis in domestic animals, (CABI Publishing, London) 2. 3 DEFRA, Salmonella in Live Stock Production in GB-22, Department of Environment, Food and Rural A.ffairs, (Veterinary Laboratory Agency, Addlestone, Surrey, UK) Taylor R 1 & Burrow M R, Survival of Escherichia coli and Salmonella in slurry on pastures and the infectivity of Salmonella Dublin for grazing calves, British Vet J, 129 (1971) Kelly W R & Collins 1 D, Potential health significance of antibiotic resistant Escherichia coli and other infectious agents present in farm effluents, in Animals and human health (CEC Eur. 69 EN) 1978, Guo X, Chen J, Brackett R E & Beuchat L R, Survival of Salmonellae on and in tomato plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening, Appl Environ Microbial, 67 (21) Guo X, van Iersel M W, Chen J, Brackett R E & Beuchat L R, Evidence of association of Salmonellae with tomato plants

7 116 INDIAN 1 EXP BIOL, NOVEMBER 24 grown hydroponically in inoculated nutrient solution, Appl Environ Microbial, 68 (22) Rasch K & Richter 1, Endemiologisches urn einen bovinen Dauerausscheider von Salmonella Heidelberg, Berliner Miichener Tierarztliches Wochenschrift, 69 (1956) eck E 1 & Hepper P T, An outbreak of Salmonella typhimurium infection associated with the spreading of slurry, Vet Record, 84 (1969) 196. lo Fett W F & Cooke P H, Reduction of Escherichia coli 157:H7 and Salmonella on laboratory-inoculated alfalfa seeds with commercial citrus-related products, J Food Protect, 66 (23) II Singh B R & Sharma V D, Isolation and characterization of Salmonella Gallinarum cytotoxic factors, Indian J Exp Bioi, 38 (2) National Advisory Committee on Microbiological Criteria for Foods (NACMCF/FDA). Microbiological safety evaluations and recommendations on sprouted seeds, lnt J Food Microbial, 52 (1999) Lang M M, Ingham B H & Ingham S C, Efficacy of novel organic acid and hypochlorite treatments for eliminating Escherichia coli 157:H7 from alfalfa seeds before sprouting, lnt J Food Microbial, 58 (2) Singh B R, Molecular characterization of cytotoxins of Klebsiella pneumoniae. Ph D thesis submitted to GB Pant Univ of Agric Techno! Pantnagar, 1997, Singh B R, Sharma V D & Chandra R, Purification and characterization of Klebsiella pneumoniae cytotoxins, Indian J Exp Bioi, 37 (1999) Singh B R & Sharma V D, Isolation and characterization of four distinct cytotoxic factors of Salmonella Weltevreden, Zbl Bakt (International J Med. Microbial), 289 (1999) Singh B R & Sharma V D, Serodetection of Salmonella Cytotoxins and toxinotyping of Salmonella strains from foods of animal origin, Indian J Comp Microbial lmmunol Infect Dis, 21 (2) Sarnish Z, Etinger-Tulczynska R & Bick M, The microflora within the tissue of fruits and vegetables, J Food Sci, 28 (1962) 259.