Morpho-molecular identification of causing anthracnose disease of banana in Pakistan.

Banana (Musa spp.) is widely cultivated as the major fruit in Pakistan. Anthracnose fruit rot caused by various Colletotrichum spp. is a serious disease. Sample of 23 banana fruits were obtained from 4 commercial orchards (43% incidence) in Uthal, Balochistan, Pakistan. One or more small light-brown to reddish-brown spots were observed on all fruits during early stage of infection and later became sunken lesion. The pieces of diseased tissues were cut from margins, surface sterilized with sodium hypochlorite (0.3%), rinsed in sterile distilled water, dried on sterile filter papers and shifted on potato dextrose agar (PDA). Colony morphology of all isolates (n=21) on PDA were white fungal colonies which became dark gray after 7 days. Smooth, aseptate, cylindrical and hyaline conidia (n=20 per isolate) were recorded and measured 10.2 to 16.1 (avg. 12.3) × 3.7 to 4.5 (avg. 3.8) µm. Genomic DNA was extracted from a representative isolate LUAWMS and sequences from the internal transcribed spacer region (ITS), β-tubulin (TUB2), calmodulin (CAL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase 1 (CHS-1) genes were amplified through polymerase chain reaction with the primer pairs ITS1/ITS4, BT1/BT2, CAL1/CAL2, GAPDH1/DAPDH2, ACTN1/ACTN2 and CHS1/CHS2, respectively. The sequences were exhibiting 100% genetic similarity with previously reported isolates SPTD26 (ITS, accession no PQ780054), PHL6, (TUB2, accession no KY475555), HNCS015 (CAL, accession no KX673577), Cer015 (GAPDH, accession no MK473911), QJ2_1 (ACT, accession no OQ613619) and CAUA43 (CHS-1, accession no KP145299) of Colletotrichum scovillei. ITS, TUB2, CAL, GAPDH, ACT and CHS-1 multi-locus sequences were deposited in GenBank under the accession numbers OK493385, OK493386, OK493387, OR449357, OR449358 and OR449359, respectively. For the confirmation of Koch's postulates, banana fruits (n=3) were surface sterilized with 70% ethanol, washed with sterile distilled water and wounded with the help of sterile needle. The spore suspension (1 × 106 conidia/mL) from LUAWMS were pipetted (10 µL) on wound fruit and incubated at 28°C with 12 h dark and light cycle. Moist paper towels was placed on bottom of a sealed crisper box and artificially inoculated fruits were shifted to a humid chamber. Three replicate boxes each contained one banana per treatment. Sterile distilled water was used as a negative control and the experiment was repeated once. After 7 days, small light-brown to reddish-brown spots (length averaged 14.9 mm, std. dev. = 4.0 mm) by 11.3 mm (std. dev. = 1.9 mm) were recorded on artificially inoculated fruits but not on control. Same pathogen was re-isolated from artificially inoculated fruit on PDA and morphological examination and molecular identified was used for the confirmation as previously described. Morphological characterization, molecular identification, multi-locus sequence analysis and Koch postulates confirmed LUAWMS as C. scovillei belongs to C. acutatum species complex (Damm et al., 2012). Previously, C. scovillei was recorded as causal organism of anthracnose disease of banana in China (Zhou et al., 2017), Watermelon in Malaysia (Goh et al., 2022) and pepper in Ontario, Canada (Ellouze, 2024) but to our knowledge, this is the first report of C. scovillei causing anthracnose of banana in Balochistan, Pakistan. The new host-pathogen association could lead to reduced banana yields, higher management costs, and potential trade disruptions due to disease spread and export restrictions.