CHAPTER 4 4.0 Results 4.

1 DNA Quality Test Nematodes extracted DNA s The quality of DNA EXTRACTED FROM NEMATODES was checked by the use of ITS R/F (Internal Transcribed Spacer Reverse/Forward) primer which s pan the ITS of most nematodes. All the nematodes samples (M. javanica TRB, M. ja vanica S.A, M. javanica 5, M. incognita S.A, M. incognita race 3, M. hapla) and a Pythium isolate used in this research generated a single 760 bp product using the ITS R/F primer (Fig 1 ). 1 8 9 2 3 4 5 6 7

Fig 1 : Amplification of the 6 Nematodes isolates using ITS R/F primer. Lane 1-9 : M. jav TRB, M. jav S.A, M. jav 5, M. inc S.A, M. inc race 3, M. hapla, Py 2 1 (control), water (-ve control) and lane 9: O GeneRuler DNA Ladder Mix (100-10000 b p ). The gel picture above shows that DNA extraction for the Nematodes isolates and a Pythium isolate was successful as the researcher managed to get distinct single expected bands for all the isolates. Thus the researcher concluded that the DNA was of good quality and can be used for the species specific PCR and genetic di versity PCR . 4.2 Checking presence of Meloidogyne species 4.2.1 MF/MR In this research the isolates used were supposed all to be in the Meloidogyne ge nus, so in the quest to indentify the nematodes that belong to the Meloidogyne g enus, MF/MR Universal Meloidogyne spp-specific primer was used to identify... ...and the results were as follows; 1 9 2 3 4 5 6 7 8

Fig 2: Amplification of the 6 Nematodes isolates using MF/MR primer. Lane 1-9: M . jav TRB, M. jav S.A, M. jav 5, M. inc S.A, M. inc race 3, M. hapla, Py 21 (con trol), water (-ve control) and lane 9: O GeneRuler DNA Ladder Mix (100-10000 bp). In all the isolates worked with in this research, a fragment of 500 bp was ampl ified in all isolates containing Meloidogyne spp. using MF/MR primers as was exp ected confirming that all the isolate used in the research belong to the Meloido gyne genus of root-knot nematodes. The 500 bp was not amplified in Pythium isola te and this shows that the MF/MR primers are highly specific for nematodes only belonging to the Meloidogyne genus and selective as the 500 bp was not amplified in the Pythium isolate a fungus. However, from the gel picture researcher saw t hat a faint band was produced in M. incognita S.A isolate. 4.2.2 MIG For checking the presence of the Meloidogyne species again a double confirmation , the MIG (Meloidogyne Incognita Group) primer which amplify DNA from M. incogni ta, M. arenaria and M. javanica, was used .and the results were as follows; 8 1 9 2 3 4 5 6 7

Fig 3: Amplification of the 6 Nematodes isolates using MIGR/MIGF primer. Lane 1-9: M. jav TRB, M. jav S.A, M. jav 5, M. inc S.A, M. inc race 3, M. hapla, Py 21 (control), water (-ve control) and lane 9: O GeneR

uler DNA Ladder Mix (100-10000 bp). The gel picture above shows that all the six Nematodes isolates belong to either M. arenaria or M. javanica or M. incognita as in all the isolates the expected 500 bp product was generated though it had some multiple bands in some isolates such as M.jav TRB, M.jav 5 and M. inc race 3. The MIG primer showed high level o f specificity as it only amplified in the nematodes samples but the 500 bp was not generated in the Pythium isolates lane 7. 4.3 Distinguishing between the Meloidogyne species The Fjav/Rjav, Far/Rar, and the Finc/Rinc primers were used in distinguishing th e Meloidogyne species. The Far/Rar primer did not amplify the expected 420 bp pr oduct as was expected in all isolates used; this might be due to the fact that t here were no M. arenaria species in the isolates used in this research. The Fjav/Rjav primer set yielded the expected band size of 720 bp (Fig 4), the e xpected for M. javanica species. 4.3.1 Fjav/Rjav 8 1 9 2 3 4 5 6 7

Fig 4 Amplification of the 6 Nematodes isolates using Fjav/Rjav primer. Lane 1-9: M. jav TRB,lane 2 M. jav S.A, lane 3 M. jav 5, M. in c S.A, M. inc race 3, M. hapla, Py 21 (control), water (-ve control) and lane 9: O GeneRu ler DNA Ladder Mix (100-10000 bp) In the above picture the expected 720 bp was detected in all samples except in M . incognita S.A which was expected as through morphological techniques this isol ate was categorised as an incognita species. However, the same band size was yie lded by other Meloidogyne incognita races and Meloidogyne hapla. This was not co nsistent with other researches published before with this primer set. This sugge sted a possible contamination in the samples. Again though the expected band 720 bp was detected, they were some multiple bands in samples like M. jav TRB, M. j av S.A and M. hapla high multiple bands being found in M. jav TRB, and this was not as expected as there was supposed to be detected single bands only. Further tests were done using the M. incognita specific primers (Finc/Rinc). The primer yielded the expected single 399 bp with M. incognita races as shown in (Fig 5). However the same band was yielded by some M. javanica races like M. javanica 5 a nd also in M. hapla but absent in the controls. This was not consistent with the expected results or other published results and suggested the root-knot nematod es samples may have been cross contaminated .

4.3.2 Finc/Rinc 1 8 Fig 5: Amplification of the 6 Nematodes isolates using Finc/Rinc primer. 2 3 4 5 6 7

Lane 1-8: M. jav TRB, M. jav S.A, M. jav 5, M. inc race 3, M. inc S.A, M. hapla, water (-ve control) and lane 8: O GeneRuler DNA Ladder Mix (100-10 000 bp). The expected 399bp band size was detected in M. jav S.A, M. jav 5, M. inc race 3 and M. hapla, but it was not detected in M. jav TRB and M. inc race. From the r esults there were some consistency and inconsistencies with other research done. That is as expected the 399bp product was detected in M. incognita race 3 which is a Meloidogyne incognita species which is supposed to be detected by Meloidog yne incognita species specific primers like Finc/Rinc primer used in this resear ch. However the 399 bp product was detected in M. javanica S.A, M. javanica 5 an d M. hapla which was not as expected as these isolates were considered not to be Meloidogyne incognita species. This brings about an element of cross contaminat ion between samples. Also there were multiple bands in M. jav S.A, M. jav 5, M. inc race 3 and M. inc SA instead of single bands only. 4.3.3 Far/Rar From the isolates used in this research there was no Meleiodogyne arenaria speci es that was detected through the use of Meleiodogyne arenaria species specific p rimers Far/Rar as the expected band was not detected in all the six isolates use d. 4.4 Determination of genetic diversity of Meloidogyne species A total of 2 Universal Rice Primers (URPs) were used in this study and all of th e primers amplified the nematodes eggs DNA producing DNA fingerprints with monom orphic and polymorphic fragments. 4.4.1 URP 17 R 1 9 Fig 6: Amplification of the 6 Nematodes isolates using URP 17 R primer. Lane 1-9: M. jav TRB, M. jav S.A, M. jav 5, M. inc S.A, M. inc race 3, M. hapla, Py 21 (control), water (-ve control) and lane 9: O GeneRu ler DNA Ladder Mix (100-10000 bp). Similarity Coefficient Fig 7: A dendrogram constructed for results obtained using URP 17 R primer The dendrogram shows the relationship between the nematodes isolates. The first point is that there is a great genetic base between the six nematodes species us ed from 0.3 to 1.0. The primer was able to cluster the nematodes isolates into 5 genetic groups as shown on the dendrogram. As shown in the dendrogram M. inc S. A and M. hapla have the highest similarity which was at around 65% hence, were t he most related isolates. The primer clustered the Pythium isolate as an outlier which was expected hence the primer proved to be effective in distinguishing be tween the isolates. 2 3 4 5 6 7 8

4.4.2 URP 38 R

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Fig 8: Amplification of the 6 nematodes isolates using primer URP 38 R. Lane 1-9: M. jav TRB, M. jav S.A, M. jav 5, M. inc S.A, M. inc race 3, M. hapla, Py 21 (control), water (-ve control) and lane 9: O ler DNA Ladder Mix (100-10000 bp). Similarity coefficient Fig 9: A dendrogram constructed for results obtained using URP 38 R primer The dendrogram shows the relationship between the nematodes isolates. The first point is that there is a great genetic base between the six nematodes species us ed from 0.0 to 1.0. The primer was able to cluster the nematodes isolates into 5 genetic groups as shown on the dendrogram. As shown in the dendrogram M. inc race 3 and M. jav 5 have the highest similarity which was at around 99% hence, w ere the most related isolates. The primer clustered the Pythium isolate as an ou tlier which was expected hence the primer proved to be effective in distinguishi ng between the isolates. Genetic diversity is of paramount importance to the plant breeders in their aim to produce resistant varieties. In this research the URP 17 R showed that the si x nematode species used were not similar, but with 38 R, M. inc race 3 and M. ja v 5 were similar this might be difficult to conclude since there was an element of cross contamination between the samples. 4.5 Validation of the protocol Results A total of five nematodes samples were used to validate the developed protocol, the samples used were obtained from the nematology section. DNA was extracted th en PCR was run using the following primers; ITS R/F, MIG, MF/MR, Finc/Rinc, Fjav /Rjav and Far/Rar. The results were as follows;

GeneRu

4.5.1 Finc/Rinc 1 6 . Lane 1-8: Sample 1, sample 2, sample 3, sample 4, sample 5, Py 21 ( control), water (-ve control) and lane 8: O GeneRuler DNA Ladder Mix (100-10000 bp). From the results it can be observed that, from the five samples used the 399 bp expected band size was detected in the first four samples which shows that there were Meloidogyne incognita species. The 399 bp band was not detected in sample five which can means that it is does not belong to the Meloidogyne incognita spe cies. 7 2 8 3 4 5

Fig 10: Amplification of the 5 unknown nematodes samples using Finc/Rinc primer

4.5.2 Fjav/Rjav

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Fig 11: Amplification of the 5 unknown nematodes samples using Fjav/Rjav primer. Lane 1-8: Sample 1, sample 2, sample 3, sample 4, sample 5, Py 21 ( control), water (-ve control) and lane 8: O GeneRuler DNA Ladder Mix (100-10000 bp). The 720 bp band size expected for Meloidogyne javanica species with Fjav/Rjav pr imer was detected in sample 1 and 2, which shows that these two samples belong t o the Meloidogyne javanica species, but the rest does not. Based on the results obtained, molecular techniques have proven to be useful app lication in the identification of root-knot nematodes species since it was able to identify nematodes from Pythium isolates and also to identify cross contamina tion in the nematode samples. Thus molecular techniques can be adapted to compli ment morphological techniques in identification of Meloidogyne nematodes.