Table 1.
List of primers used in this study.
| Sl | Primers name | Gene name (accession number) | Primers Sequences (5′–3′) | Tm (°C) | Purpose |
|---|---|---|---|---|---|
| 1 | RT miODR-1 F | Mi-odr-1 (MG780832) | GAACCACGCTCTTACGATTC | 60°C | qRT PCR |
| RT miODR-1 R | CTCCAGAAGCGACCATGTA | ||||
| 2 | RT miODR-3 F | Mi-odr-3 (MG780833) | CTGGCTATAGACCCACAGAA | 60°C | qRT PCR |
| RT miODR-3 R | GAACGTTGTCCACCTACATC | ||||
| 3 | RT miTAX-2 F | Mi-tax-2 (MG780834) | GAGGGAATATCCTGAAGCG | 60°C | qRT PCR |
| RT miTAX-2 R | CCTGATTCCACTGTTCTGG | ||||
| 4 | RT miTAX-4 F | Mi-tax-4 (MG780835) | GTGAAGTTCCTTGGCCTAT | 60°C | qRT PCR |
| RT miTAX-4 R | CAGAGCTAGCAATCATACTC C | ||||
| 5 | RT miOSM-9 F | M. incognita osm-9 (MT676864) | CATGCCTGAAGATTGGGAAG | 60°C | Cloning and qRT PCR |
| RT miOSM-9 R | GGTCAGGATTAGCACCATAC | ||||
| 6 | RT miGPC-1 F | M. incognita gpc-1 (MT676857) | CAACTTCGACAAGAGGCAA | 60°C | Cloning and qRT PCR |
| RT miGPC-1 R | CCCTATTGGTCCGGTAATTAAG | ||||
| 7 | RT mi GPA-11F | M. incognita gpa-11 (MT676854) | CGACTACGGGTATTTGTGAC | 60°C | Cloning and qRT PCR |
| RT miGPA-11 R | GAAAGTTCGGCTACGAACAG | ||||
| 8 | RT miGPA-13 F | M. incognita gpa-13 (MT676855) | GCTGAAACTTCAGAGGATGG | 60°C | Cloning and qRT PCR |
| RT miGPA-13 R | GCTGGTATTCACGAGAACG | ||||
| 9 | RT miGRK-2 F | M. incognita grk-2 (MT676858) | CTCAATTCTACGCAGCAGAG | 60°C | Cloning and qRT PCR |
| RT miGRK-2 R | GGTGCCATATAGCCAACAG | ||||
| 10 | RT miXYL-1 F | Mi-xylanase (AF224342) | GGGATTAGTTGCATACAGTTAAGATAAG | 60°C | qRT PCR |
| RT miXYL-1 R | GCTGTTCGAGTAAGCAGTAGAG | ||||
| 11 | RT miXYL-3 F | Mi-xylanase (EU475876) | GGAACTTCCGGTAAACCCTA | 60°C | qRT PCR |
| RT miXYL-3 R | GACCGGACTGATGTGTTATC | ||||
| 12 | RT miENG-1 F | Mi-β-1,4-endoglucanase (AF100549) | ACCGAGCAACTCACAAAC | 60°C | qRT PCR |
| RT miENG-1 R | GGCATTGCTACCCGTATTT | ||||
| 13 | RT miPEL F | Mi-pectatelyase (AF527788) | CGAATAACGACGAAGAGGAC | 60°C | qRT PCR |
| RT miPEL R | AGAACCGACAACCCTACA | ||||
| 14 | RT miMSP-20 F | Mi-msp-20 (AY134439) | TGGTGACGAACGCACACCTACATA | 60°C | qRT PCR |
| RT miMSP-20 R | GCGCTGTCTTTGACCATTTGCTCT | ||||
| 15 | RT miMSP-33 F | Mi-msp-33 (AY142118) | GTGGCCTCCTTTGCTTGGACATTT | 60°C | qRT PCR |
| RT miMSP-33 R | CATCACCTCCAATTACTCCGGGTT | ||||
| 16 | RT miFAT-6 F | M. incognita fat-6 (MT676846) | GAATTACTGCAGGTCCTCAC | 60°C | Cloning and qRT PCR |
| RT miFAT-6 R | CATCAGTGTCAGTCCACTTG | ||||
| 17 | RT miACS-2 F | M. incognita acs-2 (MT676837) | CCCACCAGAGGAAAGGATAA | 60°C | Cloning and qRT PCR |
| RT miACS-2 R | CGCTCCTATACCAACGATCT | ||||
| 18 | RT miMDT-15 F | M. incognita mdt-15 (MT676859) | CAGCTATGCCACCAGGTTT | 60°C | Cloning and qRT PCR |
| RT miMDT-15 R | CCTAATCCTCCTCCACCATTTC | ||||
| 19 | RT miNHR-49 F | M. incognita nhr-49 (MT676861) | GCCAATGAGGCAATTAGAG | 60°C | Cloning and qRT PCR |
| RT miNHR-49 R | CCTCGTCTACCGGTTTAGAT | ||||
| 20 | RT miELO-2 F | M. incognita elo-2 (MT676842) | CAAACGGATATTGGGTCTGG | 60°C | Cloning and qRT PCR |
| RT miELO-2 R | GGCGTGATAGGGTAGGAATA | ||||
| 21 | RT miECH-5 F | M. incognita ech-5 (MT676839) | CACTAGGTGGAGGACTAGAA | 60°C | Cloning and qRT PCR |
| RT miECH-5 R | CTTGGGAGTCTTTGAGTTCC | ||||
| 22 | RT miECH-6 F | M. incognita ech-6 (MT676840) | CGAGTCTTCTGGAAGGGATA | 60°C | Cloning and qRT PCR |
| RT miECH-6 R | GCTTAGATGGCAAAGAGGTC | ||||
| 23 | RT miFLP-12 F | Mi-flp-12 (AY804187) | TGAGGAAGCGGCCCGATAGTTCTT | 60°C | qRT PCR |
| RT miFLP-12 R | GGATGAAGAAATGCTTGGACGAGT | ||||
| 24 | RT miFLP-14 F | Mi-flp-14 (AY907829) | GCGAGTCCATGTGTAGCAGCTAAT | 60°C | qRT PCR |
| RT miFLP-14 R | GGGAGATGAAGAACGTTTACTACTTTGCC | ||||
| 25 | RT miFLP-16 F | Mi-flp-16 (EU549831) | GGCAATATTCACGAGACTGGCAAC | 60°C | qRT PCR |
| RT miFLP-16 R | GGCCATTCAATGCCTGAAGAGGG | ||||
| 26 | RT miFLP-18 F | Mi-flp-18 (AY729022) | AGGATGACTTATTGCGCCAGGA | 60°C | qRT PCR |
| RT miFLP-18 R | TTCCTTTACCGAATCTGAGCACGC | ||||
| 27 | RT miACE-1 F | Mi-ace-1 (AF075718) | CTCCTTGTTCTGAGGATTGTCT | 60°C | qRT PCR |
| RT miACE-1 R | TTTATGGAGGTGGATTCTGGAG | ||||
| 28 | RT miACE-2 F | Mi-ace-2 (AF495588) | AGATGTGGAATCCGCCTAATG | 60°C | qRT PCR |
| RT miACE-2 R | TTTACTCGGGCTCTCCTTCT | ||||
| 29 | RT miNLP-3 F | Mi-nlp-3 (KY054882) | TCCACAATTTGTGCCAGGTC | 60°C | qRT PCR |
| RT miNLP-3 R | TTGTGGGGCTGATCGTTTTC | ||||
| 30 | RT miNLP-12 F | Mi-nlp-12 (KY054885) | GATAGGCGAAAGCGGACTATT | 60°C | qRT PCR |
| RT miNLP-12 R | CAAGGGCCTGTAATCTCTCTTT | ||||
| 31 | RT mi18S rRNA F | Mi-18S rRNA (HE667742) | TCAACGTGCTTGTCCTACCCTGAA | 60°C | qRT PCR |
| RT mi18S rRNA R | TGTGTACAAAGGGCAGGGACGTAA | ||||
| 32 | RT mi actin F | Mi-actin (BE225475) | TGACTCTGGAGATGGTGTTACG | 60°C | qRT PCR |
| RT mi actin R | GTGATGACTTGACCGTCAGGC |
Table S1.
Homology parameters of unannotated genes used in this study.
| Sl | Primers name | Gene name (accession number) | Primers Sequences (5′–3′) | Amplicon length (bp) | Identity (%) | Score | E-value | Purpose |
|---|---|---|---|---|---|---|---|---|
| 1 | RT miOSM-9 F | M. incognita osm-9 homologue | CATGCCTGAAGATTGGGAAG | 150 | 100 | 124 | 3.6E‒62 | qRT PCR |
| RT miOSM-9 R | GGTCAGGATTAGCACCATAC | |||||||
| 2 | RT miGPC-1 F | M. incognita gpc-1 homologue | CAACTTCGACAAGAGGCAA | 117 | 100 | 199 | 1.4E‒18 | qRT PCR |
| RT miGPC-1 R | CCCTATTGGTCCGGTAATTAAG | |||||||
| 3 | RT mi GPA-11F | M. incognita gpa-11 homologue | CGACTACGGGTATTTGTGAC | 143 | 100 | 58 | 8.2E‒23 | qRT PCR |
| RT miGPA-11 R | GAAAGTTCGGCTACGAACAG | |||||||
| 4 | RT miGPA-13 F | M. incognita gpa-13 homologue | GCTGAAACTTCAGAGGATGG | 112 | 100 | 194 | 1.1E‒17 | qRT PCR |
| RT miGPA-13 R | GCTGGTATTCACGAGAACG | |||||||
| 5 | RT miGRK-2 F | M. incognita grk-2 homologue | CTCAATTCTACGCAGCAGAG | 124 | 100 | 202 | 4.4E‒19 | qRT PCR |
| RT miGRK-2 R | GGTGCCATATAGCCAACAG | |||||||
| 6 | RT miFAT-6 F | M. incognita fat-6 homologue | GAATTACTGCAGGTCCTCAC | 162 | 100 | 306 | 1.9E‒33 | qRT PCR |
| RT miFAT-6 R | CATCAGTGTCAGTCCACTTG | |||||||
| 7 | RT miACS-2 F | M. incognita acs-2 homologue | CCCACCAGAGGAAAGGATAA | 200 | 100 | 356 | 4.2E‒40 | qRT PCR |
| RT miACS-2 R | CGCTCCTATACCAACGATCT | |||||||
| 8 | RT miMDT-15 F | M. incognita mdt-15 homologue | CAGCTATGCCACCAGGTTT | 100 | 100 | 105 | 2.5E‒5 | qRT PCR |
| RT miMDT-15 R | CCTAATCCTCCTCCACCATTTC | |||||||
| 9 | RT miNHR-49 F | M. incognita nhr-49 homologue | GCCAATGAGGCAATTAGAG | 179 | 100 | 294 | 9.4E‒32 | qRT PCR |
| RT miNHR-49 R | CCTCGTCTACCGGTTTAGAT | |||||||
| 10 | RT miELO-2 F | M. incognita elo-2 homologue | CAAACGGATATTGGGTCTGG | 148 | 100 | 64 | 2.2E‒26 | qRTPCR |
| RT miELO-2 R | GGCGTGATAGGGTAGGAATA | |||||||
| 11 | RT miECH-5 F | M. incognita ech-5 homologue | CACTAGGTGGAGGACTAGAA | 130 | 100 | 209 | 4.7E‒20 | qRT PCR |
| RT miECH-5 R | CTTGGGAGTCTTTGAGTTCC | |||||||
| 12 | RT miECH-6 F | M. incognita ech-6 homologue | CGAGTCTTCTGGAAGGGATA | 193 | 100 | 342 | 1.3E‒36 | qRT PCR |
| RT miECH-6 R | GCTTAGATGGCAAAGAGGTC |
Table S2.
Assessment of mortality of M. incognita J2s at variable concentrations of fluensulfone and time of exposure.
| Nematode mortality (%) | |||||||
|---|---|---|---|---|---|---|---|
| Fluensulfone concentration (ppm) | 1hr | 2.5hr | 5hr | 7.5hr | 10hr | 24hr | Remarks |
| 100 | 0.86 | 3.47 | 8.62 | 18.51 | 27.78 | 100 | Reduced motility and agility observed 0.5hr onward the drug treatment |
| 75 | 0 | 1.74 | 4.35 | 8.62 | 18.18 | 100 | Reduced motility and agility observed 1hr onward the drug treatment |
| 50 | 0 | 1.74 | 8.08 | 13.79 | 13.79 | 100 | Reduced motility and agility observed 1hr onward the drug treatment |
| 25 | 0 | 0.86 | 2.59 | 8.62 | 11.01 | 90.74 | Reduced motility and agility observed 4hr onward the drug treatment |
| 10 | 0 | 0 | 0 | 2.59 | 6.03 | 75.92 | Reduced motility and agility observed 4hr onward the drug treatment |
| 5 | 0 | 0 | 0 | 1.74 | 1.74 | 43.48 | Reduced motility and agility observed 7hr onward the drug treatment |
| 1 | 0 | 0 | 0 | 0 | 1.74 | 29.57 | Reduced motility and agility in few worms observed 7–8hr onward the drug treatment |
| Vehicle control (0.05% DMSO) | 0 | 0 | 0 | 0 | 0 | 0 | No change in motility and agility |
Figure 1:
Relative fold-change expression of genes related to nematode chemosensation, GPCRs, parasitism, fatty acid metabolism, and neurotransmission in M. incognita J2s after treatment with 10, 50, and 100 ppm of fluensulfone for 5 and 10 hr. Each bar represents the log2-transformed mean ± SE (n = 3). Asterisk (*) indicates significant differential expression (*p < 0.05; **p < 0.01; ns = non-significant), analyzed by one-tailed t-test.