Articles
MOLECULAR DETECTION OF CONTAMINATING FUNGI IN LETTUCE
Article number
917_28
Pages
217 – 223
Language
English
Abstract
Bremia lactucae, Sclerotinia sclerotiorum, or Botrytis cinerea are economically important pathogens of lettuce that affect the crop in the field but can also cause damage in minimally processed salads causing high losses.
We established a fast molecular test to detect the presence of B. lactucae, S. sclerotiorum or B. cinerea in symptomless leaves of lettuce.
Two pairs of primers were designed to amplify the ITS regions of B. lactucae (one pair) and of S. sclerotiorum/B. cinerea/Alternaria sp. all together (second pair) and used in PCR reactions with DNA extracted from contaminated salads collected in commercial facilities or artificially inoculated in the lab.
Amplified products were cloned and sequenced and the sequences revealed 100% similarity with sequences of B. lactucae or of S. sclerotiorum/B. cinerea/Alternaria sp. stored in genetic databases.
Restriction digestions of the amplified PCR products with the enzymes RsaI and HindIII discriminated between S. sclerotiorum and Alternaria sp. but not between S. sclerotiorum and B. cinerea, because some isolates of B. cinerea had the sequenced ITS fragment 100% identical to the S. sclerotiorum fragment.
A multiplex PCR combining the two pairs of primers with a pair of primers amplifying in the 18S region of the lettuce, as a DNA quality control, permitted to detect simul¬taneously the presence of B. lactucae and of S. sclerotiorum/B. cinerea/Alternaria sp.
Multiplex products could also be digested with the above mentioned enzymes to discriminate some of the referred pathogens.
We established a fast molecular test to detect the presence of B. lactucae, S. sclerotiorum or B. cinerea in symptomless leaves of lettuce.
Two pairs of primers were designed to amplify the ITS regions of B. lactucae (one pair) and of S. sclerotiorum/B. cinerea/Alternaria sp. all together (second pair) and used in PCR reactions with DNA extracted from contaminated salads collected in commercial facilities or artificially inoculated in the lab.
Amplified products were cloned and sequenced and the sequences revealed 100% similarity with sequences of B. lactucae or of S. sclerotiorum/B. cinerea/Alternaria sp. stored in genetic databases.
Restriction digestions of the amplified PCR products with the enzymes RsaI and HindIII discriminated between S. sclerotiorum and Alternaria sp. but not between S. sclerotiorum and B. cinerea, because some isolates of B. cinerea had the sequenced ITS fragment 100% identical to the S. sclerotiorum fragment.
A multiplex PCR combining the two pairs of primers with a pair of primers amplifying in the 18S region of the lettuce, as a DNA quality control, permitted to detect simul¬taneously the presence of B. lactucae and of S. sclerotiorum/B. cinerea/Alternaria sp.
Multiplex products could also be digested with the above mentioned enzymes to discriminate some of the referred pathogens.
Authors
M. Mota, C. Caldeira , A.A. Monteiro
Keywords
Lactuca sativa, PCR detection, Bremia lactucae, Sclerotinia sclerotiorum, Alternaria sp., Botrytis cinerea
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