Antagonistic potential of Trichoderma strains isolated from Musa paradisiaca cv. Malnad Rasbale grown farmyards against Foc race 4 pathogen.

Foc race 4 is a causative pathogen for Panama wilt disease of Musa Paradisiaca cv. Malnad Rasbale. The cost-effective measure to control rather than the usage of agrochemicals is still not available for this cultivar. Trichoderma strains act as an antagonistic agent against different phytopathogenic fungi, including many pathogenic races of Panama wilt-causing pathogens. An attempt has been made to recognize the mode of action of this antagonistic agent in in-vitro conditions, interaction between six Trichoderma strains and Foc race 4 was �rst investigated by dual plate culture method on PDA medium. This study revealed the potential of native strain KUVKU-TH02 for the biological control of Foc race 4 pathogen affected Malnad Rasbale cultivar in in-vivo conditions rather than native isolates KUVKU-TH01, KUVKU-TV01, and KUVKU-TV02. Observations revealed the lysis of hyphal ends in inhibited colonies of the fungal pathogen. Pure culture of isolated fungal strains incubated on Potato dextrose broth made a path to isolate DNA for identi�cation and molecular characterization studies. Upon DNA sequencing native isolates sequences were deposited to NCBI genebank to gain accession IDs. The phylogenetic tree built showed the evolutionary relationship between the isolates and also the potency of native biocontrol isolates against procured isolates.


Introduction
Musa Paradisiaca cv.Malnad Rasbale (silk AAB) banana fruits are well known for their good avor and taste.It is an endemic cultivar of Malnad regions which are geographically recognized as the Central Western Ghats of Karnataka, India.The susceptibility of this cultivar to Panama wilt disease is identically high which is instigated by the pathogen Fusarium oxysporum f.sp.cubense tropical race 4 (Foc race 4) ( Prasanna et al., 2022).Panama wilt is perplexing to manage, it is one of the supreme destructive disease to all banana cultivars of India and it lasts in soil for many years, which averts the planting of banana cultivars again (Stover 1962).This pathogen can be eradicated with the application of chemicals and biocontrol agents (Ploetz 2015).Banana cultivars affected by Foc race 4 include Rasthali, Nanjanagudu Rasbale, Malnad Rasbale, Karpuravalli, Ney Poovan, Monthan, Hill banana, and Pachanadan in India (Thangavelu andMustaffa 2010, Prasanna et al., 2022).
In current years extreme practice of agrochemicals and pesticides for enhancement of plant growth and pest protection has made an impact on human health.Considerable destruction to the ecosystem, humans, and animals are caused by excessive use of chemical fertilizers and pesticides.To interchange hazardous agrochemicals, promote plant growth, and survive against pathogens many biological solutions are existing for plants through microbes.Usage of biocontrol agents like Trichoderma ecofriendly fungus is a substitute method for plant treatment against pathogens (Pilkington et al., 2010).Trichoderma, a lamentous fungus has made extensive attention worldwide (Harman and Bjorkman 2014).These species are acknowledged as biocontrol militants because of their enthusiastic attributes which include antibiosis mycoparasitism, supply of nutrient elements to plants for growth promotion, and increased nutrient and water uptake (Sharma et al., 2018 Worldwide distribution of this biocontrol agent is extraordinary and also has enlarged high biodiversity (Jiang et al., 2016).For many years most species of the genus Trichoderma have been recognized and its application for plant pathogenesis resistor and growth promotion is a success until today (Topolovec-Pintaric 2019, Harman et al., 2019).Apart from Trichoderma used as a biocontrol agent it also subsidizes enhancing soil fertility and improves the availability of nutrition in soil (Singh et al. 2018;Zhai et al. 2019;Zhang et al. 2018).Many recent investigations have revealed that native Trichoderma spp.are effective biocontrol agents, more productive, and have good biocontrol activities than arti cially induced strains because they have previously adapted to local agricultural and environmental conditions (Joshi andMisra 2013, Wang et al., 2021).Henceforth, exploration of native Trichoderma spp. is important to develop biocontrol agents for local agricultural and environmental conditions.Panama wilt of banana is a highly destructive disease in tropical countries and also in India.As biological control is an alternative method for Foc race 4 to control through Trichoderma strains, which are particularly active in plants and have been colonized near the rhizospheric part of banana plants as an active controller of the pathogen.
Our investigation aimed at evaluating the ability of four native Trichoderma strains isolated from rhizospheric parts of Musa Paradisiaca cv.Malnad Rasbale banana grown farmyards of Malnad region of Karnataka, India to check the antagonistic potential against resident Foc race 4 pathogen in in-vitro conditions and to detect the evolutionary relationships between them.

Materials and methods
2.1 Isolation of Trichoderma strains: Trichoderma harzanium and Trichoderma viride were isolated from Malnad Rasbale plants grown in farmyards near Bhadra Wildlife Sanctuary (13°43'26" N75°38'21" E), Karnataka, India.Well-grown Malnad Rasbale plant was observed and soil was collected from the rhizosphere part of the plantlet.The collected soil sample was carefully transferred to a polythene bag and instantly transferred to a thermocol container supported with ice packs and carried to the lab.For isolating fungi serial dilution method was carried out (Elad Y. et al. 1981).For acquiring pure culture, emerged hyphae were constantly subcultured on potato dextrose agar (PDA) medium.Isolated colony morphologically notifying Trichoderma strains were selected for further work.
2.2 Isolation of Foc race 4: Fresh tissue of Malnad Rasbale presenting emblematic Foc race 4 signs was collected from disease-infected farmyards of Malnad region of Karnataka, India near Bhadra Wildlife Sanctuary (13°43'26" N 75°38'21" E).Tissue samples from the pseudostem part of the pathogen-infected plant were torn vertically and the tissues were collected by cutting apart into 2-3 cm in length and were carefully transferred to a polythene bag and immediately transferred to a thermocol container supported with ice packs and was carried to the lab.Samples were then washed with running-tap water.Again samples were washed with 70% ethanol for up to 20-30sec and washed with distilled water to remove the traces of ethanol.Disinfected plant samples were then kept between sterile tissue paper to remove the outer water content.Tissue samples were inoculated on PDA under aseptic conditions and incubated for up to 72 hrs at room temperature (Venkatesh et al., 2014).
2.3 Dual-culture of Trichoderma strains against Foc race-4: For dual-culture here 9cm diameter autoclaved Petri plates were incubated with PDA, Foc race 4 was placed 2.5 cm away from the center of Petri plate followed by Trichoderma strain in opposite direction and retained under in-vitro conditions for up to 6 days at 26 °C with a photoperiod of 6-8 hours under uorescent light.After the progression in colonies growth expansion of fungi was noted to isolate the dominant Trichoderma strain for future investigations (Wang et al., 2021).Trichoderma harzianum (NFCCI-3464) and Trichoderma viride (NFCCI-2552) gained from NFCCI-Agarkar Research Institute was also used to compare the e ciency of wild strains isolated.For the investigation of two strains that showed eminent antagonistic activity was selected (Savani et al. 2020, Venkatesh et al., 2014).Observation of antagonistic action of Trichoderma against pathogen was witnessed day by day.Further, two Trichoderma strains that showed the highest antagonistic activity against pathogen were noted (Savani et al., 2020).

Molecular credentials of Trichoderma and Foc race 4:
To isolate DNA, pure fungal cultures were cultured in PDB for 6 days at 28 °C, on the 6 th day fungal mat was separated using whatsmann lter paper and ground in liquid nitrogen.Extraction of DNA from mycelia was executed using the CTAB method of DNA isolation.ITS fragments in rDNA of fungal strains were ampli ed through polymerase chain reaction (PCR) via primer pairs ITS1 (5′-TCCGTAGGTGAA CCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATAT GC-3′) (Kullnig-Gradinger et al., 2002, Samuels 2006).PCR programming for Trichoderma strains was set to 94 °C for 2 min, 30 cycles of 94 °C for 30 s, and 55 °C for the ITS fragment.For Foc race 4 initial thermal cycling, condition denaturation was made at 95°C for 3 min, followed by 35 cycles consisting of denaturation at 95°C for the 30s, annealing temperature at 55°C for 45s, and extension at 72°C for 1 min, followed by a nally 72°C for 10 min extension (Venkatesh et al., 2014).PCR products for all fungal strains were analyzed by electrophoresis on 2% (w/v) Agarose gel dissolved with Ethidium bromide (5µg/ ml) in 1xTris Borate-EDTA buffer and visioned in UV transilluminator by Alpha imager EP (Alpha Innotech Corporation, USA).The ampli ed DNA product was eluted from the gel and sequenced by Applied Biosystems DNA Analyzer 3037xl (Bio Serve Technologies, Hyderabad) (Venkatesh et al., 2014).Through ITS primers and ampli ed PCR products were achieved.BLAST algorithm helped to search for sequence resemblances.(Saitou et al., 1987, Tamura et al., 2004, Tamura et al., 2013).

Phylogenetic analysis:
The phylogenetic tree was constructed based on PCR ampli ed product, generated by ITS1 and ITS4 primers.Investigation of similar sequences was conducted through the BLAST algorithm.MEGA 11 was used to build a phylogenetic tree that detected the evolutionary relationships of the isolated fungal strains.Neighboring joining method and bootstrapping analysis for 1000 replicates were performed to detect the con dence level at the inner nodes of topology (Saitou et al., 1987;Tamura et al., 2004;Tamura et al., 2013).

Result and Discussion
3.1 DNA sequencing of Trichoderma and Foc race 4: At starting period after inoculation white hyphae appeared.Aggressive growth started after 72 hrs of incubation.After this, the whole culture turned dark green after 24 hrs.Greenish circle-like patterns were formed on PDA measuring up to 3-4 cm (Fig. 1A &  1B).Observation revealed that the strains were transparent.Conidia were spherical in shape and green in color.Observed morphology of fungi matched Trichoderma harzianum and Trichoderma viride as described in ISTH.12.10 days old inoculated infected pseudostem part on PDA (Fig. 1C), produced a white mat with mycelium again after a few days this mycelium produced a pinkish colony on PDA (Fig. 1D) (Groenewald et al., 2006).The presence of microconidia, more branches of hyphae, and sickleshaped macroconidia revealed that the isolate was Foc race 4 (Beckman 1987).
DNA isolated from mycelia executed quality DNA on 0.8% agarose gel.DNA was visualized under a transilluminator (Kullnig-Gradinger et al., 2002).DNA concentration ranged from 50-150 ng/µl.For PCR ampli cation DNA bands with outstanding molecular weight and brightness were selected.DNA sequencing was carried out.For con rmation of fungal strains, the molecular methodology was followed by cloning, sequencing through ITS (620 bp), and deposited to GeneBank by accession IDs as mentioned in Table 1.Gained ITS sequences were submitted to NCBI nucleotide BLAST.Obtained results of fungal strains revealed that they were 100% matched to Trichoderma strains.Finally, isolated fungal strains after molecular characterization revealed that they were Trichoderma harzianum, Trichoderma viride, and Foc race-4.

Table 1
Isolated Fungal strains, procured fungal strains with NCBI genebank accession ID.  5A, 5B, and 5C respectively.The evolutionary history was incidentally using the neighborjoining method.Evolutionary distances were computed by the maximum composite likelihood method.
The analysis involved 5 nucleotide sequences.Positions of codon containing gaps and data missing were eliminated.Evolutionary data analysis was performed using MEGA11 software.Molecular characterization of isolated fungal strains revealed the existence of Panama wilt causing pathogen Foc race 4 in Musa Paradisiaca cv.Malnad Rasbale and also Trichoderma strains in banana farmyards.
The evolutionary history was inferred using the Neighbor-Joining method.The optimal tree with the sum of branch lenghth = 0.02570557 is shown.The evolutionary distances were computed using the Maximum Compose Likelihood method and are in the units of the number of base substitutions per site.The analysis involved 12 nucleotide sequences.Codon positions included were 1st + 2nd + 3rd + Noncoding.All positions containing gaps were eliminated.There were a total of 509 positions in the nal data set.Evolutionary analyses were conducted in MEGA11 software (Saitou N, and Nei M 1987, Felsenstein 1985, Tamura et al., 2004, Kumar et al., 2016).
The evolutionary history was inferred using the Neighbour-Joining method.The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed.Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed.The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.The analysis involved 11 nucleotide sequences.Codon positions included were1st + 2nd + 3re + Noncoding.All positions containing gaps and missing data were eliminated.There were a total of 156 positions in the nal dataset.Evolutionary analyses were conducted in MEGA11 software (Saitou N, and Nei M 1987, Felsenstein 1985, Tamura et al., 2004, Kumar et al., 2016).
The evolutionary history was inferred using the Neighbour-Joining method.The optimal tree with the sum of branch length = 0.06658330 is shown.The evolutionary distances were computed using the Maximum Composite Likelihood method and the units are in the number of base substitutions per site.The analysis involved 7 nucleotide sequences.Codon positions included were 1st + 2nd + 3rd + Noncoding.All positions containing gaps and missing data were eliminated.There were a total of 480 positions in the nal dataset.Evolutionary analyses were conducted in MEGA11 software (Saitou N, and Nei M 1987, Felsenstein 1985, Tamura et al., 2004, Kumar et al., 2016).

Conclusion
Our investigation revealed the clear presence of Trichoderma strains, popularly used as a bio-control agent for Panama wilt disease in Musa Paradisiaca cv.Malnad Rasbale farmyards near Bhadra Wildlife Sanctuary.First-ever reporting, that this cultivar is also affected by Foc race 4 pathogen which is a common pathogen for many banana cultivars worldwide (Thangavelu andMustaffa 2010, Prasanna et al., 2022).Dual plate culture method results showed that the potency to inhibit the pathogenesis of Foc race 4 from native Trichoderma strains was high when compared to procured strains from NFCCI-Pune and here on 7th day of comparision i.e, on 168th hours of investigation revealed that KUVKU-TH02 Trichoderma harzanium strain showed the highest growth rate and NFCCI-2552 Trichoderma viride strain showed the lowest growth rate against the pathogen.This gave a clear idea for us to investigate further by using these native strains to treat Foc race 4     A: It represents the growth rate of native and procured Trichoderma harzanium strains against the Foc race 4 pathogen in cms from a time interval of 24 hrs to 168 hrs i.e. from day 1 to day 7. KUVKU-TH01 and KUVKU-TH02 are the native strains and NFCCI-3464 is the procured strain.Investigation revealed that KUVKU-TH02 showed the highest inhibition activity when compared to other strains as shown in Fig. 1E and the Graphical representation is shown below.
B: It represents the growth rate of native and procured Trichoderma viride strains against Foc race 4 pathogen in cms from a time interval of 24 hrs to 168 hrs i.e; from day 1 to day 7. KUVKU-TV01 and KUVKU-TV02 are the native strains and NFCCI-2552 is the procured strain.Investigation revealed that KUVKU-TV01 showed the highest inhibition activity when compared to other strains as shown in Fig. 1F and the Graphical representation is shown below.

Sl 1 3. 2
.sp. cubence-KUVKU-FOC01 ON334167.Inhibitory activity of Trichoderma strains against pathogen: After 72 hrs of dual culture inoculation, mycelia of Foc race-4 was surrounded by Trichoderma, and growth of pathogen was limited.Suggesting micoparasitism of the Trichoderma inhibition zone was formed as shown in Fig.1Eand 1F by native isolated Trichoderma harzanium and Trichoderma viride respectively.(Atanasova et al. 2013).When compared to reference strains wild isolated variety showed a high growth rate and inhibitory activity.When observed after 7 days of dual-culture mycelia of the pathogen was sternly suppressed by Trichoderma strains compared to procured strains from NFCCI-Pune.3.3 Phylogenetic analysis: Phylogenetic trees were constructed based on ITS rDNA sequences of Trichoderma harzanium, Trichoderma viride, and Foc race 4 strains obtained from Malnad Rasbale grown farmyards illustrating the phylogenetic relationships by intraspeci c level.The trees generated were considered because of uniformity in topology.It was constructed depending on the nucleotide sequences of isolated 5 fungal strains.The evolutionary relationship of Trichoderma strains and Foc race 4 are shown in Fig.

Figures Figure 1 A
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Figure 2 A
Figure 2