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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.
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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
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
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)
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
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
Wheat 262 germination (120 h at 25 ±1oC ) aftersoaking seed for 18 h in water containg 4.20 × 106cfu ml-1
of Salmonella enterica serovar Typhimurium (E-2391)
Black mustard (Kranti) germination (120 h at 25 ±1oC ) after soaking seed for 18 h in water containg 4.20 × 106 cfu
ml-1 of Salmonella enterica serovar Typhimurium (E-2391)
Inhibition of rooting in garlic (72 h at 25 ±1oC ) Klebsiella aerogenes (K-3173) 4.54 × 106 cfu ml-1
in soaking water
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 ±1oC and then allowed to germinate in dark on wet cheese-cloth for 36 h.
C= Control; T= Test
Temporal effect on effect of Salmonella Typhimurium on germination of Yellow mustard cultivar Yst-151
90.379.7 74.3
44.3
88.796
89.3
72
0102030405060708090
100
February May August November
Per
cen
t g
erm
inat
ion
Salmonella Control
Effect of Salmonella on germination in different seasons
Temporal effect on effect of S . Typhimurium on germination of Wheat UP-262
0
20
40
60
80
100
120
November January March May
% G
erm
inat
ion
Salmonella Control
Effect of Salmonella on germination in different seasons
Effect of Salmonella Typhimurium and Serratia fonticola on germination of Black mustard
cultivar Kranti-2 seeds in different months.
93.788.3 91.7
79.7
5547 46
9295.388
92.3
69.3
38.7
0
10
20
30
40
50
60
70
80
90
100
January February March May November
% G
erm
inat
ion
Salmonella Serratia Control
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
% G
erm
inat
ion
Salmonella
Control
Temporal effect on effect of Salmonella Typhimurium on
germination of wheat cultivar UP-2382 seeds.
91.7 89.7
43.3 44
95.7100 100
86.783
92.3
0
10
20
30
40
50
60
70
80
90
100
November December January February March
% G
erm
inat
ion
Salmonella Control
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
99.393
99.7
0
10
20
30
40
50
60
70
80
90
100
February March April
% G
erm
inat
ion
Salmonella Serratia Control
Temporal effect on effect of Salmonella Typhimurium on germination of seeds of Black mustard cultivar Varuna
42.7
26.7
71.7
92.7
263232.7
54.3
90.3
73
97.788.7 90.3
66 64.7
010
2030
405060
7080
90100
December January February March May August
% G
erm
inat
ion
Salmonella Serratia Control
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
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
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
Conclusions1.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.
ReferencesBR 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.
