Morpho-Molecular Characterization of Fusarium oxysporum F.sp. Cubense Associated Fungal Species, Antagonism and Effects on Disease Severity and Growth of Tissue-Cultured Banana Varieties

Abstract

Banana (Musa spp.), is the most produced fruit worldwide. It is a major staple food for many countries in the tropics and sub tropics serves as it serves as a food security crop and a source of income generation in countries where it is produced. Domestic consumption accounts for 85%, while international trade accounts for 15% of global banana production. However, banana cultivation is faced with many biotic and abiotic constraints. Fusarium wilt of bananas (FWB), also known as Panama disease, caused by the soil-borne fungus Fusarium oxysporum f.sp cubense (Foc) is the most important disease of bananas in the tropics and sub tropics, and Kenya is no exception. It causes yield loss of 80-100%. This study characterized the fungal microbiome of bananas from three wards in Gatundu North, Kiambu County, Kenya. A total of 612 soil samples were collected from the rhizosphere of bananas exhibiting symptoms of Fusarium wilt from the sampling sites. Ninety-eight fungal species were isolated using serial dilution on potato dextrose agar (PDA) and subsequently characterized morphologically. DNA extraction was performed using the CTAB protocol and amplification of isolate’s DNA was performed using the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) (ITS1 and ITS4, and the isolates were Sanger sequenced. The sequences were compared to the GenBank database using the Basic Local Alignment Search Tool (BLAST), and a phylogenetic tree was constructed using the neighbor-joining method in MEGA X software (v.6.1). The characterized isolates belonged to the following genera: Fusarium, Penicillium, Paecilomyces, Rhizoctonia, Trichoderma, Simplicillium, Epicoccum, Curvularia, Alternaria, Bipolaris, Exserohilum, Setosphaeria, Cochliobolus, Syncephalastrum, and several unidentified species. A second experiment was conducted to assess the antagonistic potential and mechanism of mycoparasitism of Trichoderma atroviride and Trichoderma harzianum against Foc. Both Trichoderma species were selected based on a pre-trial in vitro test of beneficial fungal species against Foc. The Dual culture technique and Culture filtrates were used to evaluate the efficacy of both Trichoderma species against Foc. Inhibition of mycelia growth of Foc was calculated using L = [(C – T)/C] x 100. Mycoparasitism of Trichoderma against Foc was determined by microscopic observation of a mixture of both fungal species taken from a 14-days old dual culture. Data of both in vitro tests were subjected to Analysis of Variance (ANOVA) and means (p<0.05) was separated using Tukey’s Honest Significant Difference (HSD) in GenStat software (v.14.0). Dual culture and culture filtrates of T. atroviride inhibited the mycelial growth of Foc by 60.56% and 65.33%, respectively. Consequently, T. harzianum exhibited 64.21% and 61.75% inhibition of Foc. As per Bell’s Degree of Antagonism, both Trichoderma species were highly antagonistic against Foc after seven days. Both species demonstrated coiling, lysis, penetration and winding as mycoparasitism. Sixty banana plantlets of four tissue-cultured banana varieties (Giant Cavendish, Grand Naine, FHIA-17 and William hybrids) were obtained from the banana nursery of the Jomo Kenyatta University of Agriculture and Technology Enterprises (JKUATES) and screened for their tolerance to Foc under five different treatments: Blank control, Negative control (Foc), Carbendazim, commercial Trianum-22 (Trichoderma harzianum, Koppert) and Trichoderma atroviride (an isolate). All treatments were applied via root dipping; except the negative control, which was applied using infested maize kernels. Infested maize kernels were also added to all pots except those of the blank control. Data on disease severity and plant growth were taken weekly for five weeks after eight weeks of inoculation of the plantlets. The means of disease severity and plant parameters were analysed in GenStat (v.14.0) software (p<0.05) and separation of means was done by Tukey’s HSD. The effects of Carbendazim, Trichoderma harzianum and Trichoderma atroviride on disease suppression and plant growth were evaluated. Disease severity for both external and internal symptoms in bananas inoculated with only Foc was significantly higher (p<0.05) than those of the other treatments. Trichoderma atroviride produced the lowest rating for both external (1.81) and internal (1.41) symptoms. All cultivars displayed both external and internal symptoms. William hybrids inoculated with Carbendazim displayed the most external symptoms (2.73), while William hybrids inoculated with T. atroviride produced the lowest rating for external symptoms (1.4). Trichoderma harzianum-inoculated FHIA-17 produced the highest internal symptoms (3.33), but FHIA-17 inoculated with T. atroviride had the lowest rating for internal symptoms (1.00). Bananas inoculated with T. atroviride displayed the lowest symptoms and were tolerant of FWB irrespective of the banana cultivars. Trichoderma atroviride improved plant height (77.94 mm), pseudostem diameter (13.83 mm), shoot biomass (11.35 mm) and root biomass (14.27 mm), while Carbendazim improved leaf width (58.34 mm) and number of leaves (6.85). In conclusion, the characterization of fungal species recovered and identified from the rhizosphere of bananas in Gatundu North, Kenya provided numerous species that are synergistic and antagonistic to Foc. Two Trichoderma species, T. atroviride and T. harzianum were effective in controlling Foc, in vitro; with both displaying various mycoparasitic activities against the pathogen. All treatments used in the greenhouse differed significantly (p<0.001) from Foc in the expression of both external and internal symptoms. Trichoderma atroviride was not significantly different (p<0.05) from the blank control in term of internal symptoms; though they differed in regard to external symptoms (p<0.05). All tissue-cultured (TC) banana plantlets that were treated with T. atroviride were more tolerant than those inoculated with the other treatments and Foc. Additionally, T. atroviride had a positive impact on plant growth (p<0.001). Hence, Trichoderma atroviride can be used as a potential biocontrol agent for the management of Fusarium wilt of banana.