Test antagonism of several Trichoderma species in suppressing the growth of Alternaria sp. in vitro

Alternaria sp. is a pathogenic fungus that causes purple blotch disease in onion plants. Losses from this disease lead to a decrease in yield by more than 70% and seed damage to 100%. For this reason, a study was conducted to test the antagonistic ability of several Trichoderma species in suppressing the growth of Alternaria sp. in-vitro as an alternative to plant disease control that is safer and more environmentally friendly. Alternaria sp. isolate. derived from onion plants located in Bale Atu, Bukit District, Bener Meriah Regency, Aceh Province. The Trichoderma species used are T. asperellum, T. harzianum, T. virens, and Trichoderma sp. Antagonism testing was carried out using a dual culture method in a 9 cm diameter petri dish. The results showed that, T. asperellum, T. harzianum, T. virens, and Trichoderma sp. able to suppress the growth of Alternaria sp. on observation of 5 HSI. The antagonistic mechanism that occurs is competition against space, nutrition, antibiosis and mycoparasites. The best percentage of resistance occurred in the treatment of T. virens, which was 77.78%. next followed by the inhibitory power of Trichoderma sp. by 72.39%, T. harzianum by 64.80%, and T. asperellum by 60.77%.

Yield loss due to purple spot disease on shallots can reach 55% [4].The damage caused by this disease causes a yield reduction of more than 70% and seed damage of up to 100% [5].The initial symptom of this disease is the appearance of small white spots on the leaves.As it progresses, the small spots enlarge to form lesions and are surrounded by a yellow zone (halo) with a purple center.Alternaria sp.usually attacks the leaves of plants, especially old leaves and can cause the tips of the affected leaves to dry [6].Weather factors such as temperature, relative humidity, and wind speed play an important role in the dispersal of Alternaria sp.daily conidia.Meanwhile, the duration of conidium germination 1297 (2024) 012066 IOP Publishing doi:10.1088/1755-1315/1297/1/012066 2 was affected by the duration of the wet leaf surface for at least four hours since the conidium Alternaria sp.attached to spring onions [7].
Generally, shallot farmers control purple spot disease by applying synthetic pesticides, because this method is considered the easiest.However, the intensive and unwise use of synthetic pesticides can have a negative impact on humans and the environment, can contaminate the soil thereby affecting soil fertility, can kill microorganisms such as worms and nitrogen-fixing bacteria in the soil [7].For this reason, it is necessary to do alternative plant disease control that is safer and environmentally friendly.One of them is by utilizing antagonistic microbes such as Trichoderma sp., Aspergillus flavus, A. niger, Penicillium sp., Rhizopus sp.[8], Pseudomonas fluorescens, Paenibacillus polymyxa [9].Various species of Trichoderma have been widely used to control plant pathogens.Trichoderma sp. is a soil saprophytic fungus that has antagonistic properties to pathogens in the form of antibiosis, mycoparasites, competition for space and nutrients [10].and can trigger plant resistance and increase crop production [11].Trichoderma sp. has several advantages such as easy isolation, broad adaptability, easy to find in planting areas, can grow quickly on various substrates, and is not pathogenic to plants [10].Another advantage of Trichoderma sp. which has high activity in producing enzymes and secreting antibiotics or volatile alkaloids to inhibit and control the growth of pathogenic fungal colonies [12].
Trichoderma sp.capable of inhibiting the growth of the pathogenic fungus Alternaria sp. on shallots up to 50.89% through an antibiosis antagonist mechanism [13].Trichoderma sp.potential as a biological control agent against purple spot disease on shallots with an inhibition of 64.55% through an antagonistic parasitism mechanism [8].And then, Trichoderma sp.capable of inhibiting the growth of the mycelia of the pathogenic fungus Alternaria sp. which comes from shallots with an inhibition power of 56% in vitro [14].Research on the use of Trichoderma sp. in inhibiting the growth of Alternaria sp.cause of purple spot disease on leek, has never been done.Currently, local isolates of Trichoderma spp.from several plants have been found.Therefore, we want to do research on the effectiveness of Trichoderma spp.local isolates from bamboo, nutmeg, coffee, and spring onions in inhibiting the growth of Alternaria sp.in-vitro.

Materials
The materials used in this study were Alternaria sp.isolates.from spring onions located in Bale Atu Village, Bukit District, Bener Meriah Regency, Aceh Province.T. harzianum isolates from the nutmeg plant, T. virens from the coffee plant, and T. asperellum from the bamboo plant were obtained from the collection of the Plant Diseases Laboratory, Faculty of Agriculture, University of Syiah Kuala, and Trichoderma sp.isolates.obtained from the soil samples of leek plantations.The materials used were 70% alcohol, 0.5% sodium hypochlorite/NaClO, potato dextrose agar (PDA) media, distilled water, tissue, chloramphenicol, plastic wrap, clear plastic, and label paper.

Isolation and Propagation of Pure Cultures of Trichoderma spp.
Soil originating from the rhizosphere of the leek plant is taken and weighed as much as 1 g, then the soil is put in a test tube containing 9 ml of sterile distilled water, so that a 10 -1 dilution is obtained.Next, the suspension was stirred using a vortex for 3 minutes.A total of 1 ml of the suspension was taken using a micropipette, then put into a test tube containing 9 ml of distilled water for a 10 -2 dilution.The suspension in the test tube is stirred again using a vortex for 3 minutes, and so on until the dilution is 10 -6 .After that, 1 ml of the suspension was taken from a 10 -6 dilution and placed in a petri dish that already contained PDA.After flattening using a spreader, the petri dish was covered with a petri cover and wrapped and then labeled and then incubated at room temperature for 7 days.Trichoderma sp.growth was inoculated repeatedly until a pure culture was obtained.For T. harzianum, T. virens and T. asperellum isolates, rejuvenation was carried out on PDA media in a petri dish.The petri dish was closed and wrapped and then labeled and incubated at room temperature for 7 days.

Preparation of Alternaria sp. pure cultures
Scallion plants that are attacked by purple spot disease are washed thoroughly with running water.After the plants are clean from the remnants of dirt that sticks, the part of the leaf that is attacked by Alternaria sp.cut into 1 cm pieces and soaked in 0.5% NaClO solution for 1 minute then soaked again in 70% alcohol for 1 minute.Then washed thoroughly with running water three times, then drained using a sterile tissue in a laminar air flow cabinet.After drying, the sections were inoculated on PDA media that had been prepared previously, and stored at approximately 28⁰C for 3 days.This work was repeated several times in a laminar air flow cabinet to prevent contamination until pure isolates were obtained.Pure isolate Alternaria sp.obtained was stored for further testing.

Antagonism test
Antagonism testing was carried out using a dual culture method in a 9 cm diameter petri dish.Alternaria sp.taken using a sterile cork borer with a size of 5 mm and placed into a petri dish, with a distance of 3 cm from the left edge of the petri dish.Trichoderma spp.taken with a size of 5 mm and placed opposite Alternaria sp., with a distance of 3 cm.The cultures were incubated at room temperature for ± 7 days.An illustration of the dual culture method is presented in Figure 1.x 100 % Information: P = Percentage of obstacles (%) r1 = Radius of the pathogen isolate away from the antagonist (mm) r2 = Radius of the pathogen isolate approaching the antagonist (mm)

Antagonist Mechanism
Observation of the antagonistic mechanism was carried out by cutting the mycelia with a 1 cm2 scalpel in the contact area of the two fungi in the dual culture test, then placing it on an object glass and observing it under a microscope.

Data analysis
Observational data were analyzed using ANOVA.The results of the F test showed a significant effect (α = 5%), followed by a different test between treatments using the Least Significant Difference Test (LSD) procedure at the 0.05 level.

Alternaria sp. Colony Growth Rate (mm/day)
Measurement of the growth rate of Alternaria sp.colonies.determined based on the increase in colony diameter per day.The results of analysis of variance showed that the growth rate of Alternaria sp.due to double test with Trichoderma spp.significant effect at 1 day after inoculation (DAI) and very significant effect at 2 to 9 DAI.The average growth rate of Alternaria sp. both in control and as a result of treatment with Trichoderma spp., can be seen in Figure 2  Based on Figure 2 above, the growth of the Alternaria sp.mycelium.in the double test treatment with Trichoderma spp.slower than the control.This is caused by the growth of the mycelium of the fungus Trichoderma spp.more competitive so it grows faster to fill the petri dish.At 6 DAI, the growth of pathogenic colonies was inhibited by antagonist agents.When viewed from the growth rate of Alternaria sp.every day, it can be seen that Alternaria sp. also experienced an increase in diameter, from the beginning of growth to the 9 day, but not as fast as the growth of Trichoderma spp.Alternaria sp. began to grow to fill the petri dish on the 9th day of observation.The research conducted by Sudewi stated that, the time needed for A. porri to grow to fill the petri dish is ±14 days [16].
T. virens isolate had the highest inhibition rate against Alternaria sp.compared to Trichoderma spp.other.The results of the analysis of variance at 2 to 5 HSI showed that the ability of T. virens was very significantly different from the other three Trichoderma species in the ability to inhibit the growth of Alternaria sp colonies.T. virens acts as a good competitor for nutrients and space to grow.This indicates that the secondary metabolites produced by T. virens are capable of inhibiting the growth of pathogenic colonies.One of these secondary metabolites is the viridin compound which can inhibit the germination of pathogenic fungal spores [17].The activity of the chitinase and glucanase enzymes produced by Trichoderma can also inhibit the growth of pathogenic fungi [18].
The growth rate of antagonistic fungi (which is determined by fast colony growth), is an important indicator of biological agents.An important characteristic of antagonist agents is that they have faster colony growth than pathogens, so they can inhibit the growth of pathogens [19].The average growth rate of Alternaria sp. this will later be related to the high and low percentage levels of inhibition of Trichoderma spp.against Alternaria sp.If the average growth rate of pathogenic fungi is low, then the inhibition percentage of antagonistic fungi is high.
One of the factors that influence the high or low percentage of inhibition of pathogenic fungi is the growth rate of antagonistic fungi [20].Pathogenic fungi whose growth was inhibited were thought to be due to inhibition by Trichoderma spp.through mycoparasitism, antibiosis, and competition.The growth rate of antagonistic fungi is thought to be an indicator of competition for space and nutrients between antagonistic fungi and pathogenic fungi [21].

Inhibition of Trichoderma spp. against Alternaria sp.
Measurement of the percentage of inhibition was determined based on measuring the radius of the pathogen colony away from the antagonistic fungus colony (r1), and the radius of the pathogen colony approaching the antagonistic fungus colony (r2) with an interval of observation every 24 hours.The results of the analysis of variance showed that the four isolates of Trichoderma spp. at 1 HSI no significant effect, but very significant effect at 2, 3, 4 and 5 HSI.The average percentage of inhibition of Trichoderma spp.against Alternaria sp. can be seen in Figure 3.
Based on Figure 3, it can be seen that all Trichoderma spp.isolates tested had positive inhibition against Alternaria sp. with varying percentages of inhibition, namely T. asperellum (60.77%), T. harzianum (64.80%), T. virens (77.78%) and Trichoderma sp.(72.39%).T. virens isolate showed the best inhibitory ability, namely 77.78%.This proves that, T. virens is able to dominate living space and can compete against pathogens.T. virens was able to suppress the growth of hyphae Alternaria sp. even to the extent that the T. virens mycelium overgrowth over the pathogenic colonies (overgrowth) (Figure 4c).This overgrowth may be due to the growing nature and rapid sporulation of T. virens itself.
Susanna et al. stated that T. virens had a better ability to suppress Lasiodiplodia theobromae (a cause of dieback in nutmeg tree) compared to T. asperellum and T. harzianum [22].While another study, T. asperellum has a greater ability to inhibit the growth of pathogens compared to T. harzianum.The difference in inhibition illustrates the ability of each Trichoderma spp.isolate.to inhibit the growth of pathogens [14].Various studies have shown that Trichoderma has different inhibitory and antagonistic activity against Alternaria sp. even between the same Trichoderma species.This difference is thought to be influenced by the type, amount, and quality of the antibiotics or other substances produced by Trichoderma spp.which can inhibit the growth of pathogens.The success of Trichoderma spp. in inhibiting Alternaria sp. is largely determined by the inhibition mechanism of Trichoderma spp itself and also the secondary metabolites it produces [23].Secondary metabolites of Trichoderma spp.can be in the form of antibiotic compounds, enzymes, toxins and hormones [17].Trichoderma produces secondary metabolites that are antibiotics such as viridiol, azaphilon, peptaibol, and peptaibiotics [24], enzyme compounds such as chitinase and glucanase enzymes [18].
If the percentage of inhibition of antagonistic fungi against pathogens is <60% of the surface of the petri dish, then the antagonistic fungi have a minimal inhibitory effect, but if the inhibition percentage is > 60%, then the antagonistic fungi are said to be able to maximally inhibit the growth of the pathogen [25].Based on this opinion, it can be said that the four Trichoderma spp.tested were able to inhibit the growth of pathogenic Alternaria sp.colonies.

Antagonist Mechanism
The success of Trichoderma spp. in inhibiting Alternaria sp. is largely determined by the antagonistic mechanism of the Trichoderma species itself.Therefore, observations were made on the condition of the colony and the morphology of the pathogenic hyphae after being tested for antagonists with Trichoderma spp.On the first day of observation after inoculation, there was no antagonistic mechanism between the two tested fungi.On the second day, the growth of the two fungi approached each other so that the growth of the pathogenic fungus was inhibited.
Macroscopically, the mechanism of inhibition by Trichoderma spp.occurs through antibiosis, and competition.There is competition between Trichoderma spp. with Alternaria sp.seen in the growth of Trichoderma spp.isolates.which is faster than the growth of Alternaria sp., which then causes Trichoderma spp.can fill the space more quickly so that the growth of Alternaria sp.become obstructed (Figure 4).Competition occurs because these two microorganisms require limited amounts of nutrients and space.Nutrients needed such as carbohydrates and glucose.This is reinforced by the opinion of Asniah et al. (2021) who stated that the growth rate of antagonistic fungi is thought to be an indicator of competition for space and nutrients between antagonistic fungi and pathogenic fungi [21].
Based on its ability to inhibit the growth of Alternaria sp., the fungi T. virens and Trichoderma sp.act as good competitors for space and nutrients so that at 6 DAP, these two fungi lacked space to grow Inhibition (%) and resulted in Trichoderma colonies growing above the pathogen (Figures 4c and 4d).This overgrowth also suggests that there has been a mechanism of hyperparasitism and mycoparasitism.According to Abdel-Rahim and Abo-Elyousr, mycoparasite interactions are morphologically characterized by antagonistic mycelium overgrowth in pathogenic mycelia and this overgrowth is caused by the growth and sporulation properties of Trichoderma [26].The of antibiosis occurs in the treatment of Alternaria sp. with T. harzianum (Fig. 4b), seen from the mycelium of Alternaria sp.The growth was not mixed with T. harzianum mycelium, and a clear zone was formed as a dividing zone for the growth of the two fungi.The mechanism of antibiosis can also be seen from the color change on the PDA media which turns yellowish.This is in accordance with the opinion of Ainy et al. (2015) that, the mechanism of antibiosis is indicated by the formation of a clear zone at the junction of the mycelium of antagonistic fungi and pathogenic fungi [27].Apart from looking at the clear zone, the mechanism of antibiosis can be seen by the presence or absence of color changes in the media due to antibiotic compounds produced by the fungus [28].The antibiosis mechanism is indicated to occur due to the presence of secondary metabolites produced by T. harzianum, both in the form of antibiotics, toxins, enzymes and hormones so that they can inhibit the growth of pathogens.The mechanism of antibiosis can occur due to the presence of secondary metabolites produced by the fungus T. harzianum as a defense for the fungus to survive or compete.The clear zone was formed due to the presence of secondary metabolites produced by T. harzianum in the form of alametichin, paracelsin, and trichotoxin [27].
Microscopic observations were also carried out to observe the presence or absence of mycoparasite mechanisms and whether there were changes in the hyphae of the pathogen Alternaria sp. after being tested antagonist with Trichoderma.The mechanism of mycoparasites is indicated by the presence of hyphae of T. asperellum, T. virens, and Trichoderma sp. which is wrapped around the hyphae of Alternaria sp. (Fig. 5b, 5d, and 5e).Trichoderma hyphae initially grow elongated, then entangle the pathogenic hyphae and then penetrate the pathogenic hyphae.This causes changes in the hyphae structure of Alternaria sp.such as hyphae Alternaria sp. which undergo lysis, and malformations in the form of abnormal enlargement of the hyphae, swelling at the ends of the terminal hyphae, wrinkling and shrinkage of hyphae, growth of circular hyphae, and discoloration of hyphae.
When antagonistic fungi which are mycoparasites reach their host, the hyphae then wrap around or squish the host hyphae by forming a hook-like structure [29].Along with the stabbing of the hyphae, this mycoparasite fungus secretes enzymes such as chitinase and β-1-3 glucanase enzymes which will degrade the cell wall of the pathogenic fungus.As a result, the hyphae of the pathogenic fungus will be damaged, the protoplasm will come out and the fungus will die [18].
Trichoderma can penetrate the host cell wall with the help of certain enzymes and use the contents of its host's hyphae as a food source and cause mycolysis.Mycolysis is the loss of protoplasm in the fungus cell wall structure.This mycolysis causes a number of symptoms, such as swelling, shortening and lysis of cell walls and results in abnormal growth of hyphae.Morphological changes in hyphae vary and are specific between the Trichoderma species used and are thought to be buried in their ability as antagonists and growth inhibitors [27].The morphological appearance of pathogenic hyphae is presented in Figure 5 below.Hyphae of Alternaria sp. which underwent lysis were found in interactions with T. harzianum, T. asperellum, and T. virens (Figures 5c, 5d and 5e).T. virens also causes hyphae of Alternaria sp.experience swelling at the terminal ends of the hyphae to form a chain of beads (Figure 5e).Degraded hyphae and abnormal changes in shape are an indication of the activity of antibiotic compounds or enzymes released by antagonistic fungi.Degradation of fungal cell walls is mainly caused by chitinase, glucanase and protease.Chitinase is an enzyme that is able to break down chitin, so that Trichoderma spp.can degrade the cell walls of Alternaria sp. which contains chitin.Glucanase is an enzyme that can hydrolyze glucan compounds.Glucans are found in many fungal cell walls [30].
T. harzianum is known to be able to produce a number of secondary metabolites in the form of alametichin, paracelsin, trihotoxin which can destroy mold cells by damaging the cell membrane.Apart from that, T. harzianum can also produce the enzymes chitinase, laminarinase, β-3-glucanase which can cause cell wall lysis.T. asperellum is able to produce enzymes that can cause lysis of its host hyphae and has mycoparasitic properties that can inhibit the development of pathogens [31].Important secondary metabolites from T. asperellum are tetradecanoic acid (myristic acid), 1,2-benzenecarboxylic acid [32], 2H-Pyran-2-one and diethyl phthalate [33].Apart from that, T. asperellum also produces alkaloids and saponins [17].
The wrinkled and shrunken hyphae were found to be the effect of mycoparasites from Trichoderma sp.(Figure 5b) and changes in pigmentation were only found in Alternaria sp. who were exposed to T. virens.This change in pigmentation is thought to be caused by T. virens absorbing nutrients from the hyphae of Alternaria sp.so that the hyphae look empty and hyaline, which then causes the hyphae to die and their growth is hampered.The interaction between pathogens and antagonists causes the color of pathogen hyphae to change to become clear and empty because the cell protoplasm is used by biocontrol agents as nutrients [34].Meanwhile, according to Inayati et al. (2019), volatile compounds produced by T. virens cause morphological changes in pathogen hyphae such as changes in hyphal color (reduced or excessive pigmentation).The volatile compounds produced by T. virens are sesquiterpenes, azulene, β-elemene, α-gurjunnene, caryophillen, isoledene, muurolene, cadinene, (3R)-3-Phenyl-2,3dihydro-1H-isoindol-1-one , and 3-Octanone [35].
T. virens is a mycoparasite and produces antibiotics in the form of gliotoxin, gliovirin and viridiol which are fungistatic.The antibiotic compounds produced can also inhibit the development of pathogens, parasitize pathogens with direct penetration and are also faster in using oxygen, water and nutrients such as glucose and carbohydrates so that they are able to compete with pathogens [36] 4. Conclusions T. asperellum, T. harzianum, T. virens, and Trichoderma sp.able to suppress the growth rate of A. porri colonies at 5 DAP.The highest percentage of inhibition occurred in the T. virens treatment, namely 77.78%, followed by Trichoderma sp.amounted to 72.39%, T. harzianum amounted to 64.80%, and T. asperellum amounted to 60.77%.The antagonistic mechanisms that occur are competition for space, nutrition, antibiosis, and mycoparasites.Further research needs to be done on the use of Trichoderma spp. in controlling Alternaria sp. on a field scale.

Figure 1 .
Figure 1.Illustration of a dual culture model between Alternaria sp. and Trichoderma spp.

2. 6
Alternaria sp.colony growth rate (mm/day) Colony growth rate was observed every day until the control petri dish (without Trichoderma sp.) was filled with the fungus.Measurements are measured using a formula that refers to Crueger et al. (1984) which is modified as follows: rate (mm/day) d2 = Increase in diameter of Alternaria sp.colonies.next day(mm) d1 = Increase in diameter of Alternaria sp.colonies.previous day(mm) t2 = The next observation day (day) t1 = Day of previous observation (day) 2.7 Inhibition of Trichoderma spp.against Alternaria sp.(%) Growth inhibition of the mycelium of the fungus Alternaria sp. by the fungus Trichoderma spp.calculated based on the Fokkema formula (1973), namely:

Figure 2 .
Figure 2. The average growth rate of Alternaria sp.colonies.(mm/day).The numbers followed by the same letters are not significantly different based on the least significant difference test (LSD) at the 0.05% level.

6Figure 3 .
Figure 3. Percentage of inhibition of Trichoderma spp.against Alternaria sp.The numbers followed by the same letters are not significantly different based on the least significant difference test (LSD) at the 0.05% level.

Figure 5 .
Figure 5. Hyphae morphology of Alternaria sp.(400x magnification) .Hiphae morphology of Alternaria sp.(400x magnification) ;(A) Size of Alternaria sp.hypha, normal.(B) Hypha of Trichoderma sp.wrapped around the hypha of Alternaria sp. and the hypha experience shrinnkage.(C) Hypha of Alternaria sp. which is lysed due to interaction with T. harzianum.(D) Hypha of T. asperellum wrapped around hypha of Alternaria sp.so that the hypha undergo lysis.(E) Hypha of T. virens wrapped around hypha of Alternaria sp.so that the hypha lyse and swell and the hypha became hyaline (arrow).