Pathogens of Public health importance with zoonotic potential also affect the germination and rooting in plants. Germinating seeds of different crops are affected to varying degree and all are not equally susceptible. Cultivars also modulate the susceptibility of seed germination to different pathogens. Crop production may be affected in soil contaminated with sewage and night soils. Effect of bacteria on germination of seeds also modulated by month/ season of sowing.

1. Germinating seed model: an
alternative of animal models for
bacterial toxins and zoonotic
pathogens
Dr. BR Singh
Head Division of Epidemiology
Indian Veterinary Research Institute, Izatnagar
&
Director CCS NIAH, Baghpat

2. Disease models are needed for...
• For determining pathogenic potential of bacterial isolates
in veterinary and medical diagnostic laboratories.
• For understanding pathogenesis
• For development of vaccines
• For evaluation of new methods for diagnosis and
intervention techniques for clinical use I
• It is not always feasible to study a disease in host because
of:
– Ethical problems
– Bisafety
– Availability and economy
– Regulatory constraints

3. In vivo Models for toxins of
microbes
• Rabbit ligated ileal loop assay, closed mouse
and infant mouse assay, monkey and kitten
tests for enterotoxins.
• Infant mouse, dogs, monkeys, rats and guinea
pigs for Neurotoxins.
• Rabbit skin, rabbit eye, guinea pig eye, guinea
pig skin for irritant, dermotoxins and necrotoxins.
• Nematode, molluscs and many other animals
have been used. (limulus lysate assay for
endotoxin)

4. In-vitro techniques
• Tissue culture tests (Vero cells, Caco-2,
MDBK, MDCK, Y1 adrenal, CHO cell etc.)
• Immunological methods: Biken test,
Nagler’s reaction, RIA, ELISA, FAT, CFT
• Probes (hot and cold)
• PCR and RT-PCR
• Chemical analysis: FPLC, HPLC, Atomic
absorption spectrophotometery

5. Why more models?
• For better understanding of pathogenesis,
• For more economic and simple methods
• For better adaptability of the models to
poorly equipped labs
• For academic purpose

6. Wheat 262 germination (120 h at 25 ±1o
C ) after
soaking seed for 18 h in water containg 4.20 × 106
cfu ml-1
of Salmonella enterica serovar Typhimurium (E-2391)

7. Black mustard (Kranti) germination (120 h at 25 ±1o
C ) after
soaking seed for 18 h in water containg 4.20 × 106
cfu
ml-1
of Salmonella enterica serovar Typhimurium (E-2391)

8. Inhibition of rooting in garlic (72 h at 25 ±1o
C )
Klebsiella aerogenes (K-3173) 4.54 × 106
cfu ml-1
in soaking water

9. Germination of maize (Ganga) seeds after exposure to
Salmonella Cytotoxin I @1mg/ 100 ml of soaking water.
Seeds were soaked over 18 hr at 25 ±1o
C and then allowed
to germinate in dark on wet cheese-cloth for 36 h.
C= Control; T= Test

10. Temporal effect on effect of Salmonella Typhimurium on
germination of Yellow mustard cultivar Yst-151
90.3
79.7
74.3
44.3
88.7
96
89.3
72
0
10
20
30
40
50
60
70
80
90
100
February May August November
Percentgermination
Salmonella Control
Effect of Salmonella on germination in different seasons

11. Temporal effect on effect of S . Typhimurium on germination of Wheat UP-262
0
20
40
60
80
100
120
November January March May
%Germination
Salmonella Control
Effect of Salmonella on germination in different seasons

12. Effect of Salmonella Typhimurium and Serratia
fonticola on germination of Black mustard
cultivar Kranti-2 seeds in different months.
93.7
88.3
91.7
79.7
55
47 46
92
95.3
88
92.3
69.3
38.7
0
10
20
30
40
50
60
70
80
90
100
January February March May November
%Germination
Salmonella Serratia Control

13. Temporal effect on effect of Salmonella Typhimurium on
germination of lentil cultivar PL-5 seeds.
0
10
20
30
40
50
60
70
80
90
100
February May September November
%Germination
Salmonella
Control

14. Temporal effect on effect of Salmonella Typhimurium on
germination of wheat cultivar UP-2382 seeds.
91.7 89.7
43.3 44
95.7
100 100
86.7
83
92.3
0
10
20
30
40
50
60
70
80
90
100
November December January February March
%Germination
Salmonella Control

15. Temporal effect on effect of Salmonella
Typhimurium and Serratia fonticola on
germination of Black mustard cultivar P-Mutant
seeds.
74
92.7 90
56.7
92.3
75.3
99.3
93
99.7
0
10
20
30
40
50
60
70
80
90
100
February March April
%Germination
Salmonella Serratia Control

16. Temporal effect on effect of Salmonella Typhimuriumon
germination of seeds of Black mustard cultivar Varuna
42.7
26.7
71.7
92.7
26
3232.7
54.3
90.3
73
97.7
88.7 90.3
66 64.7
0
10
20
30
40
50
60
70
80
90
100
December January February March May August
%Germination
Salmonella Serratia Control

17. S.
No.
Variety of seeds (botanical name)-cultivar Average percent germination (standard deviation)
Salmonella
yphimurium
Serratia
fonticola
Control 1 Control 2
1 Bell Pepper (Capsicum annuum)-Calfornia Wonder 55.0 (1.0) 74.7 (3.1) 84.7 (2.5) 84.3 (1.5)
2 Black Gram (Cicer arietinum)-BG-256 63.7 (2.3) NT 64.0 (6.2) 65.3 (3.2)
3 Black Gram (Cicer arietinum)-C-235 93.3 (2.1) NT 98.7 (1.2) 97.7 (1.6)
4 Black Gram (Cicer arietinum)-PG-186 55.0 (2.6) NT 58.0 (1.0) 58.3 (1.2)
5 Black mustard (Brassica nigra)-BSH-1 54.3 (0.6) 55.0 (1.0) 91.7 (1.5) 92.3 (1.4)
6 Black mustard (Brassica nigra)-JMM-927 90.7 (1.2) 76.3 (1.5) 93.0 (2.6) 93.7 (2.1)
7 Black mustard (Brassica nigra)-Karishma 70.3 (1.5) 82.7 (3.1) 99.3 (1.2) 98.7 (2.2)
8 Black mustard (Brassica nigra)-Kranti-2 88.3 (1.5) 46.0 (2.0) 88.0 (1.0) 89.3 (1.4)
9 Black mustard (Brassica nigra)-Krishna-1 69.0 (2.0) NT 72.7 (4.5) 72.3 (2.5)
10 Black mustard (Brassica nigra)-NUDB-09 70.3 (0.6) 67.0 (2.0) 70.3 (1.5) 71.7 (1.3)
1 Black mustard (Brassica nigra)-P-mutant 74.0 (3.6) 56.7 (2.1) 99.3 (1.2) 96.7 (1.6)
12 Black mustard (Brassica nigra)-RH-45 94.3 (0.6) 93.3 (1.5) 93.3 (0.6) 94.7 (1.6)
13 Black mustard (Brassica nigra)-RH-7846 90.0 (1.0) 75.3 (0.6) 99.7 (0.6) 98.3 (1.3)
14 Black mustard (Brassica nigra)-RK-9501 99.3 (1.2) 85.3 (1.2) 99.7 (0.6) 97.7 (2.6)
15 Black mustard (Brassica nigra)-Varuna 71.7 (1.5 54.3 (2.1) 88.7 (1.5) 89.7 (1.3)
Effect of Salmonella and Serratia on germination
of seeds of different crops and varieties

18. 16 Cowpea (Vigna unguiculata var sinensis)-UPC-607 73.3 (1.5) NT 98.0 (1.0) 97.7 (1.5)
17 Cucumber (Cucumis sativus)- Lucky 97.7 (0.6) 98.7 (1.5) 99.0 (1.0) 99.3 (1.4)
18 Guar, or clusterbean, (Cyamopsis tetragonoloba) 93.3 (4.2) 96.7 (2.1) 98.7 (0.6) 99.7 (0.3)
19 Kakari (Cucumis melo var. utilissimus)-Green long Spru 77.7 (1.5) 90.7 (1.2) 90.7 (3.1) 94.7 (2.1)
20 Knol-Knol (Brassica oleracea var. gongylodes)-Winter Green 55.0 (1.0) 85.0 (2.6) 99.0 (1.0) 98.3 (1.8)
21 Lentil (Lens esculenta)-PL5 39.0 (3.6) NT 18.0 (1.0) 18.3 (1.8)
22 Lentil (Lens esculenta)-PL-62 98.3 (1.5) NT 96.0 (1.0) 96.7 (1.3)
23 Maize (Zea mays)-Ganga 71.0 (1.7) 95.0 (1.0) 95.0 (1.0) 94.3 (1.5)
24 Maize (Zea mays)-JK Surbhi 78.7 (1.2) 76.7 (0.6) 98.3 (1.5) 98.3 (0.6)
25 Maize (Zea mays)-Naveen 87.0 (1.0) 86.7 (1.5) 86.7 (1.5) 87.7 (1.7)
26 Maize (Zea mays)-Suraj 90.3 (1.5) 89.3 (3.5) 90.7 (1.2) 91.0 (1.6)
27 Mung (Pahseolus aureus)-Asha 97.3 (1.2) 95.3 (1.5) 92.7 (3.5) 93.3 (2.5)
28 Mung (Pahseolus aureus)-ML-818 98.7 (1.5) 99.0 (1.7) 100.0 (0.0) 99.0 (1.0)
29 Mung (Pahseolus aureus)-Pant Mung-3 73.3 (3.2) 98.3 (1.2) 99.0 (1.0) 100.0 (0.0)
30 Mung (Pahseolus aureus)-Pant Mung-5 98.7 (1.2) 95.3 (1.5) 99.7 (0.6) 98.7 (0.6)
31 Musk melon (Cucumis melo var. reticulatus)-Kazari 78.7 (2.1) 25.0 (1.0) 58.3 (1.5) 59.3 (1.4)
32 Okra (Abelmoschus esculentus)-Large green 59.0 (1.0) 44.7 (2.5) 79.7 (1.5) 78.3 (2.5)
33 Onion (Allium cepa)- Large red 50.3 (5.5) 29.3 (5.1) 84.7 (3.1) 86.7 (4.1)
34 Pumpkin (Cucurbita pepo)-Large 63.7 (5.8) 41.3 (3.2) 82.0 (2.0) 82.7 (2.6)
35 Radish (Raphanus sativus)-White 93.3 (1.2) 99.7 (0.6) 99.3 (1.2) 97.3 (1.2)
Effect of Salmonella and Serratia on germination of seeds of
different crops and varieties

19. 36 Sem beans (Phaseolus vulgaris)- Beuaty 99.0 (1.0) 99.0 (1.0) 99.7 (0.6) 99.3 (0.5)
37 Sweet Peas (Pisum sativum)-Pant P-5 91.7 (1.5) 98.7 (1.2) 99.0 (1.0) 97.0 (1.6)
38 Tomato (Lycopersicon esculentum)-Choice-21 86.0 (1.0) 90.3 (1.5) 99.3 (1.2) 99.0 (1.0)
39 Tomato (Lycopersicon esculentum)-T-27 90.7 (1.5) 89.3 (1.5) 93.0 (0.0) 92.0 (1.0)
40 Water melon (Citrullus lanatus)-Honey dew 100.0 (0.0) 98.0 (1.0) 99.0 (1.0) 96.0 (3.0)
41 Wheat (Triticum aestivum)-BPW-175 95.0 (1.0) NT 98.3 (0.6) 97.0 (2.6)
42 Wheat (Triticum aestivum)-BPW-299 95.0 (1.0) NT 94.7 (2.1) 94.0 (2.0)
43 Wheat (Triticum aestivum)-BPW-343 99.7 (0.6) NT 100.0 (0.0) 99.3 (1.2)
44 Wheat (Triticum aestivum)-BPW-373 99.7 (0.6) NT 99.3 (1.2) 100.0 (0.0)
45 Wheat (Triticum aestivum)-BPW-502 100.0 (0.0) NT 99.7 (0.6) 98.0 (0.0)
46 Wheat (Triticum aestivum)-Raj-3765 99.3 (1.2) NT 99.7 (0.6) 99.0 (0.0)
47 Wheat (Triticum aestivum)-UP-2382 95.7 (1.5) 90.0 (1.0) 92.3 (1.2) 95.7 (1.2)
48 Wheat (Triticum aestivum)-UP-262 21.7 (1.2) 90.0 (1.0) 93.0 (1.0) 93.3 (1.7)
49 Yellow mustard (Sinapis alba)-Yst-151 90.3 (1.5) 32.0 (2.0) 88.7 (1.5) 89.7 (1.7)
Effect of Salmonella and Serratia on germination of seeds of
different crops and varieties

20. Conclusions
1.Pathogens of Public health importance with zoonotic
potential also affect the germination and rooting in plants.
2.Germinating seeds of different crops are affected to
varying degree and all are not equally susceptible.
3.Cultivars also modulate the susceptibility of seed
germination to different pathogens.
4.Crop production may be affected in soil contaminated
with sewage and night soils.
5.Effect of bacteria on germination of seeds also
modulated by month/ season of sowing.

21. References
BR Singh, Mudit Chandra, Ravi Kant Agrawal & Babu Nagrajan. 2007. Interactions between Salmonella enterica subspecies
enterica Serovar Typhimurium and Cowpea (Vigna unguiculata variety Sinensis) seeds, plants, and persistence in hay.
J. Food Safety. 28:169-187. 10.1111/j.1745-4565.2007.00070; Url: http://www3.interscience.wiley.com/cgi-bin/fulltext/118494253/PDF
BR Singh, Ashvani Kumar, Sugandh Agrawal and Anita Verma. 2007. Effect of Salmonella Typhimurium and Serratia
fonticola on germination of seeds of common crops. Journal of Infection in Developing Countries (ISSN 1972-2680)
August 2007 Vol. 1, No. 1: 67-71. Url: http://www.oloep.org/uploadedFiles/jidc/issues/2007_08/12_ms1307_singh_F.pdf.
BR Singh, Mohan Singh, Preetam, Babu N, Mudit Chandra and Ravi Kant Agarwal. 2006. Prevalence of multidrug-resistant
Salmonella on ready-to-eat betel leaves (Paan) and in water used for soaking betel leaves in North Indian cities. J. Food Protection.
69:288-292. http://dx.doi.org/10.1043%2F0362-028X%282006%29069%5B0288%3APOMORB% 5D2.3.CO%3B2
BR Singh, Preetam Singh, Anita Verma, Sugandh Agrawal, Babu N, Mudit Chandra and Ravi Kant Agarwal. 2006. A study on
prevalence of multi-drug resistant (MDR) Salmonella in water sprinkled on fresh vegetables in Bareilly, Moradabad, and Kanpur
(Northern Indian cities). Journal of Public Health. 14 (3): 125-131. Doi: 10.1007/s10389-005-0015-3;
Url: www.springerlink.com/index/67620UP624047122.pdf
BR Singh, Mudit Chandra and Ravikant Agarwal. 2005. Interaction of Salmonella enterica Subspecies enterica Serovar Typhimurium
and Mung Bean (Phaseolus aureus) Plants. J. Food Protection. 68 (3): 476-481.
Url: http://dx.doi.org/10.1043%2F0362-028X%282005%29068%5B0476%3AIOESST%5D2.3.CO%3B2
BR Singh, Mudit Chandra, Ravikant Agarwal and Babu, N. 2005. Curing of Salmonella enterica serovar Typhimurium
contaminated cowpea seeds and sprouts with vinegar and chlorinated water. J. Food Processing and Preservation. 29:
268-277. DOI: 10.1111/j.1745-4549.2005.00027.x. Url: Url: http://www3.interscience.wiley.com/cgi-
bin/fulltext/118698667/PDFB R Singh, Ravikant Agarwal & Mudit Chandra (2004). Pathogenic effects of Salmonella enterica subspecies enterica serovar
Typhimurium on sprouting and growth of maize. Indian J. Exp. Biology, 42:1100-1106.
Mahtab Z. Siddiqui, B.R. Singh, R.K. Agarwal and S.K. Srivastava. 2006. An in vitro seed model for determination of Salmonella
serovars toxicity. Ind. J. Comp. Microbiol.Immunol.Infect.Dis. 27(1), 42-43.