DETERMINATION OF ANTIBIOSIS OF Trichoderma species AGAINST FUNGI ASSOCIATED WITH CORN

Biological control of food spoilage microorganisms is gaining more attention because it is a safe and cheap technique. This study evaluated the antagonistic potential of Trichoderma species against corn spoilage fungi by plate co-culture technique for seven days. Fungal isolates from corn spoilage were examined morphologically and microscopically. The percentage growth inhibition (PGI) of Trichoderma species against the corn spoilage fungi were A . flavus (15%), A . niger (14%), A . terreus (62.9%), Fusarium spp. (5.9%), Nigrospora spp. (61.4%) and Penicillium spp. (62.5%). The result obtained in this study revealed that Trichoderma spp. had significant inhibitory effects against the growth of fungal pathogens associated with corn spoilage. Therefore, it could be explored for control of post-harvest fungal spoilage of corn. It is recommended that in order to compare the antagonistic strength of the Trichoderma species, different species of Trichoderma should be tested against the same spoilage fungi.


INTRODUCTION
Maize (Zea mays L.) is one of the most important cereal crops worldwide, ranking first with a total global production of over 1.15 billion tons (Oli Awoke, 2023).It is an important oily and economic crop and has been utilized as human food and animal feed (Kaul et al., 2019).However, maize is highly susceptible to fungal spoilage and consequently mycotoxin contaminants, including aflatoxins (AFs) (Mendoza et al., 2017).Biological control is defined as the reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state, by one or more organisms accomplished naturally or through manipulation of the environment or host or antagonist or by mass introduction of one or more antagonists.Biological control is found to be both environmentally and economically sound (Dukare et al., 2019: Zhang et al., 2021a).Various biocontrol agents such as fungi and bacteria have been identified for the control of postharvest diseases of many fruits and play an important role in sustainable agriculture and management of plant pathogens (Dukare et al., 2019;Mukherjee et al., 2020;Morales-Cedeno et al., 2021;Zhang et al., 2021a;Abd-Rabboh et al., 2021;Yassin et al., 2022).It has also been investigated that the potential of antagonistic microorganisms to control decay of fruits and vegetables depends on their ability to colonise fruit surfaces and adapt to various environmental conditions (Dukare et al., 2019).Biological control using antagonistic microbes to control postharvest disease and decay caused by pathogens is nowadays an emerging and attractive option.The use of antagonist microbes in postharvest disease is advantageous over synthetic fungicides in that antagonist microbes do not produce toxic residues, environmentally friendly, safer application method, easy to deliver and economical to produce (Zhang et al., 2021a) .The study aimed to determine the inhibitory activity of Trichoderma species against fungi associated with post-harvest spoilage.Its objectives were;(i) Isolation and identification of Trichoderma species from soil sample.(ii) Isolation and identification of spoilage fungi from corn.(iii) Antibiosis test of Trichoderma species against isolated spoilage fungi.

Sample collection
Fresh corns (Zea mays L.) were purchased from two traders in Choba Market.The corns were kept for five days until sings of fungal spoilage was seen.Then the spoilt parts of each corn were cut for isolation of the spoilage organisms.Topsoil of farmland in University of Port Harcourt was collected for the isolation of Trichoderma species.

Isolationand identification of Trichoderma species from soil
A modified method of isolation and identification of Trichoderma spp. as demonstrated by Siddiquee (2017) was adopted.Soil sample of 10g was dissolved in 100mL of 0.1 % (w/v) Tween 20 sterile solutions and re-suspended by agitation at 150rpm and room temperature for 20min.The suspension was left on the bench of laminar flow (aseptically covered flask or tube of suspension) for 10min to decant insoluble particles and four serial dilutions was carried out with 1mL aliquot in 9mL of distilled water.From these solution (10 -3 and 10 -4 ), 1mL aliquots were seeded in Petri dishes with Trichoderma selective agar medium (TSM) by the pour plate method.The plates were incubated at 28ºC for 4-7 days until appearance of green fungal colonies showing Trichoderma features.These potential Trichoderma colonies were sub-cultured in Petri dishes containing Potato Dextrose Agar (PDA) medium and incubated at 28ºC for 7 days.Trichoderma strains were identified by analysis of morphology of colonies and visualization of structures of conidiophores with microscope.

Isolation and identification of corn spoilage fungi
A modified method of isolation and identification by Madbouly et al. (2023) was adopted.The corn samples with fungal growth were surface-sterilized using 70% ethanol for 30s and a 1% NaOCl for 3min and washed 3 times with distilled water.The samples were cut into small pieces (1cm 2 ) with a sterile scalpel, mashed using a sterilized laboratory mortar and pestle.Then 1g of the mashed corn was diluted in 9mL distilled water, ImL of the dilution was placed on potato dextrose agar (PDA) plates, supplemented with 150µgmL -1 of streptomycin to inhibit bacterial contamination and spread out using a sterile spread rod.After incubation for 72-96h at 28ᵒC, the emerging fungal hyphae from the segments were picked up onto new plates and then purified using the single spore technique.The pure fungal cultures were kept on PDA slants at 4ᵒC for further studies.

Antibiosis test of Trichoderma species against spoilage fungal isolates
Antibiosis (antagonistic activity) of isolated Trichoderma species against isolated fungal pathogens of corn were tested in vitro following the dual culture/co-culture method of Ferreira et al. (2020).A sterile cork borer (diameter 10mm) was used to transfer test fungus (pathogen) onto two sterile PDA plates halfway between the centre and edge of the plates.Then the antagonist (Trichorderma sp.) was transferred with the sterilized cork borer onto one of the plates halfway between the centre and edge the plate, opposite the test fungus.Same set up was done for all the pathogens isolated.Then the culture plates were incubated for seven (7) days at 28ᵒC, colony growth of both biocontrol agent (Trichoderma sp.) and pathogen were observed constantly and the radial growth of the pathogens in the control plates and dual culture plates (in the direction of the antagonist Trichoderma sp.) were recorded daily up to the seventh (7 th ) day (day 7) after inoculation, the percentage inhibition of radial growth of pathogens (antagonism of Trichoderma sp.against pathogens) was calculated using the formula below.P. G. I (%) = C−F C × 100 (Gwa and Ekefan, 2017) Where: P.G.I = Percentage growth inhibition.C = the distance (mm) from the point of inoculation to the colony margin of control plate on day 7 after inoculation.F = the distance (mm) of fungal growth from the point of inoculation to the colony margin in the treated plate in the direction of the antagonist (Trichoderma sp.) on day 7 after inoculation.

RESULTS AND DISCUSSION
The result aspresented in
Vesicles are hemispherical and phialides cover the entire surface and are produced from a primary row of metulae.
Hyphae are small and septate and give rise to phialides that produced singlecelled microconidia.
Hyphae are hyaline and septate and produced brush-like conidiophores.

Pc
Two concentric rings with green conidial production.
Key: Pc = Plate count  2022) also reported coculture of Trichoderma harzanium and A. terreus for removal of organic pollutant.The antagonism observed in this study could be a situation of strains specific inhibition.Trichoderma spp.showed the very little antagonism (PGI 5.9%) against Fusarium spp.This result does not agree with the results by Abhiram and Masih (2018) when they cocultured on plate Trichoderma viride and Fusarium oxysporum, and the results revealed that Trichoderma viride showed maximum inhibition 71.0% over Fusarium oxysporum strain (E) and minimum inhibition 62.50% over Fusarium oxysporum strain (D).Anjum et al. (2020) using the dual culture technique evaluated antagonistic potential of T. atroviride, T. hamatum, T. harzianum, T. longibrachiatum, and T. viride against Fusarium oxysporum f. spp.capsici and the result was 67.18%, 70.15%, 68.75%, 69.46% and 66.75% inhibition, respectively, which does not agree with the result in this study.Trichoderma spp. was antagonistic to Nigrospora spp. in this study at a percentage growth inhibition of 61.4%.This result corroborates with the report by Zhang et al. (2021b) who reported relative inhibitory rate of 89.80% by Trichoderma spp.against Nigrospora sphaerica.Trichoderma spp.displayed a high antagonism against Penicillium spp. at a percentage growth inhibition of 62.5%.This result agrees with the antagonism of 35.5±1.9% reported by Yuliantoro and Prihatiningrum (2023).

CONCLUSION
The result obtained in this study revealed that Trichoderma spp.antagonized many genera of corn spoilage fungi.Therefore, it could be explored for control of post-harvest fungal spoilage of corn.The use of Trichoderma species as biocontrol agents can reduce the use of chemical fungicides which can have negative impacts on the environment and human health.Further studies are needed to evaluate the efficacy of Trichoderma species under field conditions and to develop practical applications for their use in corn production.

table 1
The result revealed that Fusarium spp (Pc4) grew well despite the presence of Trichoderma spp.whileAspergillus niger (Pc3) and Penicillium spp.growths were inhibited by Trichoderma spp.The result as presented in Figure (1) and (2) shows the antagonism of Trichoderma spp.against corn spoilage fungi.The result revealed that Trichoderma spp. was most antagonistic against Aspergillus terreus (62.9%), followed by Penicillium spp.(62.5%) and the least antagonism was on Fusarium spp.(5.9%).