1. Development of a specific detection assay to investigate growth and survival of BCAs in the field & Field trials of a potential BCA in Vietnamese rice paddies during a crop season in Ô Môn (Can Tho, Vietnam)

2. Problem (1) Fungus, Rhizoctonia solani, causes sheet blight in rice.
R. Solani presents high infection rates; able to destroy up to 50% of the crops in infected fields.
10% of the worlds annual rice production is discarded because of sheet blight caused by R. solani produced
R. solani presents sclerotia formation  sclerotia can survive (stay dormant) in the fields for 2 years without water.

3. Problem (2)
Until now rice fields infected with R. solani have been treated with fungicides, although fungicides have little effect R. solani
Negative effects of excessive use of pesticides on the environment and human health have become increasingly clear
Various R. Solani strains cause sheet blight and roth in maize, carrots and potatoes.

4. Solution
Solution; an environmentally stable and cheap approach to combat R. solani
This project propose; the use of biological control agents (BCAs) to combat R. solani. The approach has the advantage of being both environmentally friendly and sustainable.

5. Finding a potential BCA
A common soil bacteria complex, the Burkholderia cepacia complex (BCC) was known to exist in rice fields already infected with R. solani
BCCs found in the Mekong River Delta (Can Tho, Vietnam) have been isolated and screened for hyphae association
Two bacterial strains, the B. vietnamiensis VL-16.5 and AG-7.3, show promising biological control abilities against R. solani
Figure R. solani: ~90h (p.i.) with BCAs. Upper left shows TG-17.2, upper right shows AG-10.3, lower left corner shows AG-7.3 and lower right corner shows VL-16.5.
4.16 F. graminearum: ~90h (fungus) and ~68h (bacteria) (p.i.) with BCAs. Upper left shows TG-17.2, upper right shows AG-10.3, lower left corner shows AG-7.3 and lower right corner shows VL-16.5.

6. Finding unique genetic fingerprints(1)
RAPD-PCR performed: random amplification of polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random.
Figure . The random primers are tested on a: TG-17.2, b: VL-16.5, c: donated lysate. PCR was run with the RAPD-PCR setup and with the use of a 1.5% agorose-gel. Distinct patterns are seen for some of the primers. Especially 270, 272 and 287 should be noticed. A 2 log ladder was included as marker.

7. Finding unique genetic fingerprints (2)
Selecting primers and finding unique sequences for AG-7.3 and VL-16.5

8. Analyzing the fragments
> B.Vi.Frag.03 (500 bp final sequence) CGGGTTGGTCCGGCTCAACAAAAGCGGCTCGCCTACGCCCAGAAAAACA AGATTCTGGACGCGTTCAAGCGCAAGTTGCGCAATGGGGAAGATTTGATG GCCCGGTCCGTCAGTTCGGAATTGGCGAAGCGCTTCAACGTGTCGCAAA GTCACGTCCGTGAGCTTCGAAAGAACTGGTTGAGAACGTTAAAACCTGAC AAACGCAACTGACGAAACCACAGTTGCGTTTGTGAAATCGTAAACGCAGT TGTCGATACGACAGTTGCGTTTCAGCCTGCATGCTTCGCGTTATCCCGTG CAAACCCGCACGGTCCCATAACTAGCGGAGCATAGATGCAAAACACGTC GTACCCGATCAATCGCGTTGTGCGCATGCGCTGCCTCCTCGGCCGCGTC GGTCTGAGCAAAAGCGAAATCTACCGACGCATTCAGGCAGGCGCGTTCC CGAAGCCTATTCCGCTCGGCA
Primer Number
Primer orientation
Primer Length (bp)
Tm /oC
G/C content
Primer Sequence
Product size (bp) 1
Forward 20
60.13
45.00
CAATGGGGAAGATTTGATGG
194
Reverse
60.06
50.00
GCTGAAACGCAACTGTCGTA 2
59.89
GACAAACGCAACTGACGAAA
238
60.10
CCTGAATGCGTCGGTAGATT 3
162
59.86
GATCGGGTACGACGTGTTTT 4
60.20
ACCACAGTTGCGTTTGTGAA
219

9. Making and testing the specific primers
Figure . 3 Burkholderia strains run with real-time PCR setup and the 4 primer pairs returned from DNA Technologies. A 1.5% agarose gel was used for electrophoresis. A 2 log ladder was included for evaluation of the fragments. The setup gave the following result: primer 1 and 4 yielded bands for AG-7.3 and related strains, where as primer 2 and 3 only produced bands for AG-7.3.
Figure 4.10 Primer 2 and 3 added in a real time-PCR setup with a larger variety of soil bacteria. A 1.5% agarose gel was used for gel electrophoresis. The figure shows that primer 2 and 3 only produce bands for B. vietnamiensis strain AG-7.3. Note the formation of primer dimer below the ladder.

10. During Real-Time PCR

11. Microcosm trials

12. Fields Trials in Vietnam

13. Results from field trial
Data is still being analysed
7% decrease in infection rate, from the first year