Exploring the antagonist potential of indigenous Trichoderma spp., Bacillus, and Pseudomonas against Phytophthora palmivora of Soe mandarin in East Nusa Tenggara, Indonesia

Phytophthora palmivora-induced root and basal stem rot pose a significant threat to the survival of Soe mandarin plants in East Nusa Tenggara Province, Indonesia. We isolated four Trichoderma species, five Bacillus, and 13 Pseudomonas isolates from the rhizosphere of healthy Soe mandarin. This study pursued two main objectives: (a) assessing the inhibitory capabilities of indigenous Trichoderma spp., Bacillus, and Pseudomonas towards P. palmivora in vitro, and (b) investigating the combined efficacy of Trichoderma spp., Bacillus, and Pseudomonas in controlling P. palmivora in a pot trial. Results revealed that four Trichoderma species (T. asperellum, T. hamatum, T. harzianum, and T. viride) significantly inhibited the growth of P. palmivora. Notably, T. asperellum exhibited the highest inhibition, at 84.31%, followed closely by T. harzianum (84.11%), T. viride (83.67%), and T. hamatum (80.28%). Bacillus and Pseudomonas exhibited varying levels of inhibition to P. palmivora growth in vitro, with the most substantial inhibition observed in Bacillus 1, followed by Pseudomonas 6. In the subsequent pot trial, the application of Trichoderma, Bacillus, or Pseudomonas, either individually or in combination, significantly increased the height increment and reduced the disease incidence caused by P. palmivora in Soe mandarin seedlings.


Introduction
Soe mandarin is an essential agricultural product in West Timor, East Nusa Tenggara province in Indonesia.The fruit is spherical and porous, with orange flesh and very sweet.Ripe fruit skin is yellow and reddish with a smooth, oily skin surface and is very easy to peel because the flesh is separated from the skin.Soe mandarins have been a significant profit source for growers in West Timor's upland since the seventies.These mandarins were important regarding money and as part of the traditional agricultural system.
Historically, this Soe mandarin was planted close to households in an assorted cropping system with other horticultural and perennial crops.However, the authorities subsequently initiated grafting as IOP Publishing doi:10.1088/1755-1315/1302/1/012017 2 an additional reproduction technique to replace the ancient technique of directly planting seeds to increase the harvest [1].
The initial record of root and basal stem rot disease affecting Soe mandarin caused by Phytophthora spp.dates back to 1998.Subsequently, Mudita (2013) stated that diseases on Soe mandarin have caused many trees to die at comparatively early stages, for both the flourishing and the non-flourishing plants, from around two to nine years old [1].Various research studies have indicated that P. citrophthora, P. nicotianae, and P. palmivora were responsible for the basal stem rot disease in Soe mandarin [2,3].Simamora et al. 2018 also determined that P. palmivora was the causative agent of root and stem rot in Soe mandarin.This disease can lead to significant fatalities, mainly when a plant is attacked by multiple pathogens [4].
The regional authorities in West Timor have declared that they have formulated their top attempts to defend Soe mandarins from pests and disease infection, but the decline continues.Most farmers do disease control by using the California mixture as suggested by the authorities.The California mixture is recommended for diseases caused by fungi [5] but not very suitable for diseases caused by pseudofungi, such as Phytophthora.The chemical mixture intended to be used as a protectant on vigorous plants was discovered to be used on very poorly plants, or even if it was applied to healthy trees, it was quickly washed away by rain [1].Because of that, there is a need for other more effective control techniques for Phytophthora.
Ecological safety and foodstuff protection have achieved much respect globally everywhere.Consequently, biology and ecology-safe pest management tools are now being broadly implemented.Various fungi and rhizobacteria are recognized to have an antagonistic ability as opposed to phytopathogens.Pseudomonas and Bacillus rhizobacteria are among the genera categorized as biocontrol agents.In addition, the genus Trichoderma has good characteristics for restricting disease caused by phytopathogens such as Phytophthora, Fusarium, and Sclerotium, among others [6].
The general disease control technique using biocontrol agents relies on exploiting a solitary microorganism for disease control.Nonetheless, relying on a solitary biocontrol agent raises doubt about consistently achieving antagonistic effects against phytopathogens [7].To address this inconsistency, combining two biocontrol agents or two different types of biocontrol agents simultaneously is recommended.
Much work has demonstrated that integrating more than one biocontrol agent to manage phytopathogens gives better results than applying only one.This is attributed to the synergistic effect resulting from the combination of various biocontrol agents, incorporating antagonistic mechanisms like competition and the synthesis of inhibitory compounds [9][10][11][12].The possibility of treatment of two or more biocontrol agents for controlling disease caused by Phytophthora in Soe mandarin has not been investigated.Consequently, this study aimed to (a) assess the antagonistic ability of indigenous Trichoderma spp., Bacillus, and Pseudomonas against P. palmivora in vitro, (b) examine the antagonistic effect of combined Trichoderma spp., Bacillus, and Pseudomonas in controlling P. palmivora in a pot trial.

Microorganisms used and culture media conditions
Trichoderma asperellum, T. hamatum, T. harzianum, T. viride, Bacillus, and Pseudomonas were sourced from the root zone of disease-free Soe mandarin plants.In addition, P. palmivora was isolated from the soil and basal stem rot of diseased Soe mandarin trees.All these microorganisms were isolated in Tubuhue Village in South Central Timor District in 2017 [4].Intermittently, Trichoderma spp.and P. palmivora were transferred to a Potato Dextrose Agar (PDA) medium.Bacillus and Pseudomonas were preserved on a Nutrient Agar at 25 ± 2°C.

In vitro antagonism test of P. palmivora by Trichoderma spp.
Trichoderma asperellum, T. hamatum, T. harzianum, and T. viride were used in this trial.Each isolate of Trichoderma and P. palmivora isolate was individually introduced to PDA medium and incubated at 25 ± 2°C for four days.Trichoderma mycelium fragments (0.5cm 2 ) were then positioned along one edge of the Petri plate.In contrast, mycelium fragments (0.5cm 2 ) of P. palmivora, obtained from actively growing colonies, were placed on the opposite edge of the Petri plate.The Petri plates were subsequently placed on a laboratory bench at a temperature of 25 ± 2°C for a duration of seven days.The colony diameter of P. palmivora was measured on a daily basis.The percentage of inhibition in P. palmivora circular development caused by Trichoderma was analyzed as follows: The percentage of inhibition (%) = (R1-R2)/R1x 100.R1 represents P. palmivora circular development in the control Petri dish, and R2 denotes P. palmivora circular development in the presence of Trichoderma in the trial Petri dish.The analysis of variance (ANOVA) test was performed to determine the percentage of inhibition, and subsequently, the least significant difference (LSD) test was conducted.

In vitro antagonism test of P. palmivora by Bacillus and Pseudomonas
Phytophthora palmivora plug (0.5cm 2 ) was placed at the centre of the Petri dish on NA medium.The rhizobacterial culture (Bacillus or Pseudomonas) was taken using a loop and streaked in a straight line just before and behind the P. palmivora plug.The distance between P. palmivora and rhizobacteria was 2 cm.As a control, P. palmivora was grown separately in an NA medium without rhizobacteria.The Petri dishes were incubated at ambient temperature and monitored every day until P. palmivora in control entirely covered the Petri plate.There were three replicates for this trial.The colony diameter of P. palmivora was measured on a daily basis.The proportion of inhibition of P. palmivora circular growth by rhizobacteria was calculated using the following equation: The inhibition percentage (%) = (R1-R2)/R1 x 100.R1 corresponds to P. palmivora circular development in the control Petri plate; while R2 signifies P. palmivora circular development towards the rhizobacteria in the trial Petri plate.An analysis of variance was used to examine the percentage of inhibition, and further assessed using the LSD test.

Preparation of Trichoderma, Bacillus, and Pseudomonas pellet
A Trichoderma and rhizobacteria pellet bioformulation was prepared according to Simamora et al. 2018 [4].First, the carrier material is weighed 1:1:1 (100 g: 100 g: 100 g), then placed in a heatresistant plastic, homogenized, and sterilized.The Trichoderma formulation was prepared: the cooled carrier mixture was mixed with 120 mL of Trichoderma suspension, 150 mL of egg white, and 150 mL of coconut oil and then mixed until smooth and can be formed.The rhizobacteria formulation was prepared: the cooled carrier mixture is mixed with 120 mL of rhizobacteria suspension, 150 mL of egg white, and 150 mL of coconut oil and then mixed until smooth and can be formed.For the formulation of a mixture of Trichoderma and rhizobacteria, the cooled carrier mixture is mixed with 60 mL of Trichoderma suspension, 60 mL of rhizobacteria suspension, 150 mL of egg white, and 150 mL of coconut oil, then mixed until smooth and can be formed.The pellet's diameter was 3 cm, and it weighed 2.8 g.

Antagonistic test of Trichoderma spp., Bacillus, and Pseudomonas and their combinations in inhibiting P. palmivora in the pot trial
Based on the positive results of individual applications of four Trichoderma species and 18 rhizobacterial isolates in earlier in vitro trials, their combined effect in controlling the P. palmivora was performed in the pots.Before carrying out a pot trial, the activities of the isolates to be combined were evaluated in vitro.All species of Trichoderma were compatible, but only three Trichoderma spp.were used in this pot trial because they were also found to be compatible with Bacillus 1, Pseudomonas 3, and Pseudomonas 6.
In vivo growth inhibition test of P. palmivora was carried out on healthy Soe mandarin seedlings grown in sterile soil in pots.The rootstock was obtained from certified Phytophthora-free seed breeders to guarantee that the plants are free from P. palmivora.The seedlings used in the test were three months old.This study was organized utilizing a fully randomized design, incorporating 16 treatments with three replications.The treatments were: (A) P. palmivora only as control, (B) T. The number of pellets given to Soe mandarin seedlings in polybags was four pellets/pot, given two weeks before the application of P. palmivora.Each pot was given four pellets of Trichoderma spp. or Bacillus or Pseudomonas if the treatment was individual application.Two pellets of Trichoderma and two of Bacillus (or Pseudomonas) were given per polybag for the combination treatments.Phytophthora palmivora was applied by placing 20 mL of P. palmivora suspension in a polybag.The suspension was obtained by mixing 20 mL of sterile distilled water with the pure P. palmivora culture in a Petri dish containing PDA.The age of pure P. palmivora culture is seven days.The suspension was mixed evenly and applied to pots.
After carrying out the application according to the treatments, observations were made on the symptoms of Soe mandarin seedlings.Observation begins one day after the application process.Alterations in plant height were assessed by measuring them before and after applying experimental treatments.Additionally, disease incidence was documented.Statistical analysis involving ANOVA was conducted to analyze seedling height increment and disease incidence, followed by the LSD test.

In vitro antagonism test of P. palmivora by Trichoderma spp.
The level of inhibition exerted by Trichoderma spp.against P. palmivora exhibited a highly significant difference.According to the 5% LSD results, T. asperellum demonstrated greater effectiveness, resulting in the highest percentage of inhibition against P. palmivora.However, this was not significantly different from the inhibition observed with T. viride and T. harzianum (Table 1).The lowest inhibition percentage was achieved by T. hamatum, but in general, all Trichoderma species tested had very high antagonistic abilities (above 80%) against P. palmivora (Figure 1A).Trichoderma spp.functions as a biocontrol agent by applying four distinctive methods to impede pathogens' growth.These methods include competing for food in the shared growth environment, utilizing antibiosis to release toxic biochemichal compounds to suppress pathogens, employing mycoparasitism by attaching to and binding with pathogenic fungal hyphae, and penetrating pathogenic fungal hyphae while synthesizing cell wall-degrading enzymes [13,14].
One of the best methods for finding beneficial fungi that are effective against the growth of specific pathogens is to look around for healthy plants with many diseased plants or disease outbreaks [15].Given this background, in this investigation, four Trichoderma species were obtained from the soil and fine roots of healthy Soe mandarin trees in areas affected by P. palmivora root and basal stem rot disease.
Trichoderma was initially isolated from soil in 1974 and has since proven to be a recognized biocontrol asset [16].This research aligns with previous findings indicating that Trichoderma spp.can reduce the development of Phytophthora spp. in both laboratory and field conditions.Sriwati et al. 2015 [17] reported that Trichoderma isolated from Aceh Sumatra reduced the wounds on cocoa husks and saplings caused by Phytophthora.Moreover, the combination of Trichoderma spp.and potassium fertilizer extended the incubation time and decreased P. palmivora specks on cocoa leaves by as much as 75.37% [18].In a different review, Choudhary et al. 2021 [19] highlighted the effective management of citrus gummosis/foot rot and maximized harvest using T. asperellum as a soil treatment.

In vitro antagonism of P. palmivora by Bacillus and Pseudomonas
The analysis of variance revealed a significant effect on the growth inhibition of P. palmivora in the tested rhizobacterial isolates (Figure 1B).The average inhibition percentages of P. palmivora and the LSD test by 18 rhizobacterial isolates are displayed in Table 2. Table 2 reveals that all tested rhizobacteria effectively restrained the development of P. palmivora, with Bacillus 1 exhibiting the highest inhibition percentage, followed by Pseudomonas 6 and Pseudomonas 3.
The colonies of rhizobacteria effectively restrained the development of P. palmivora, primarily because of the prolonged growth of P. palmivora in the medium.No growing hyphae were in the inhibition zone, and it looked immaculate.This condition indicates that the possible metabolite compounds produced by these bacteria can decrease the growth of P. palmivora.Cauler et al. [20] emphasized that Bacillus and Pseudomonas bacteria are abundant in soil and plants, existing and demonstrating multipurpose antagonistic actions as opposed to pathogens.Bacillus and Pseudomonas are recognized as suitable applicants for a biocontrol method because of their prevalence in diverse ecological, resilience, and persistence capabilities.Furthermore, they also deliver a variety of bioactive substances [21].The majority of these bioactive substances consist of secondary metabolites that exert detrimental effects on pathogens, directly or indirectly influencing processes such as antibiosis, competition, plant growth stimulation, and defense [22].

Antagonistic test of Trichoderma spp., Bacillus, and Pseudomonas and their combinations in inhibiting P. palmivora in the pot trial
The results of P. palmivora inhibition tests by Trichoderma, Bacillus, and Pseudomonas and their combinations in the pot trial showed varied results.All the plants, including those in the control group, inoculated with P. palmivora survived without fatalities.Nevertheless, the control plants that were solely inoculated with P. palmivora exhibited symptoms of gummosis (Figure 1C).Plants treated with Trichoderma, Bacillus, and Pseudomonas and their combinations showed no disease symptoms (Figure 1D), but the plant height increment varied (Table 3).This result shows that all the antagonistic microorganisms could inhibit the growth of P. palmivora, but their inhibitory abilities varied.Table 3 indicates that the treatment with T. asperellum resulted in the highest plant increment, though it did not show a significant difference from the T. asperellum + Pseudomonas 3 treatment.Subsequently, the treatments with Pseudomonas 3, T. hamatum + Pseudomonas 3, Bacillus 1, T. harzianum, and T. harzianum + Bacillus 1 followed in ranking.This study also underscores that whether applied individually or in combination, Trichoderma, Bacillus, and Pseudomonas can impede the expansion of P. palmivora and reduce disease incidence.Trichoderma spp. is the most widespread fungi in the soil and has an antagonistic effect on fungal pathogens.Besides its wide-ranging adaptability, Trichoderma can release toxins that suppress or execute other fungi.The high susceptibility of P. palmivora to Trichoderma spp. was also validated by Yao et al. 2023 [23].They mentioned that the effectiveness of T. asperellum could be attributed to its direct penetration in the sporocysts, coiling around Phytophthora hyphae, the formation of appressoria on the top of hyphae causing damage, and the secretion of hydrolytic enzymes like laminarinase.La Spada et al. [24] observed in their research that both T. asperellum and T. atroviride demonstrated a significant reduction in root and crown rot disease severity in tomatoes, primarily induced by Phytophthora nicotianae.
The hostile Pseudomonas spp.use their antipathogen influence by inducing fungal membrane damage, resulting in cytoplasmic leakage.Furthermore, Pseudomonas spp. release lytic enzymes and antifungal compounds that result in the perforation and lysis of fungal hyphae.Bacillus spp.produced lipopeptides that interacted with fungal membranes, causing the fungal pathogen to shrink and distort [25].The findings of this study align with research conducted in central India, affirming that Trichoderma, Bacillus, and Pseudomonas effectively managed gummosis, and root and basal stem rot in citrus [26].The application of Trichoderma as a bio-inoculant exhibits a significant outcome that can be useful to plants, such as the alteration of the rhizospheric microorganisms.Trichoderma's settlement of root and rhizosphere significantly adjusts the number and variety of local bacterial inhabitants, particularly within bacteria [27].Soil-dwelling rhizobacteria interact with plants, by suppressing plant infections through antagonistic activities and stimulating plant growth [28].The outcomes of this study also indicate the potential utilization of beneficial microbial consortia, such as Trichoderma and rhizobacteria, for plant protection.Niu et al. 2020 [29] and Collinge et al. [30] explain that the cooperative action of beneficial microbes can enhance rhizosphere colonization and the control of soil-borne diseases.Additionally, Poveda and Eugui [27] determined that the combined actions of Trichoderma and bacteria offer more significant advantages than the individual contributions of each, indicating their potential as a viable option for crop management and disease or pest control in contemporary agriculture.

Conclusions
The utilization of indigenous Trichoderma spp., Bacillus, and Pseudomonas rhizobacteria, obtained from the soil of healthy Soe mandarin trees, has exhibited potential efficacy to control the root and basal stem rot disease attributed to P. palmivora.This work underscores the efficacy of indigenous Trichoderma spp., Bacillus, and Pseudomonas as a viable alternative to the unselective use of chemicals in agroecosystems.Future investigations should focus on applying Trichoderma, Bacillus, and Pseudomonas in the field.Additionally, identifying secondary metabolites produced by these microorganisms is imperative for advancing our comprehension and facilitating further development in this field.

Figure 1 .
In vitro and pot trial results.A. Comparison growth between Trichoderma and P. palmivora in the Petri dish, B. Comparison growth between rhizobacteria and P. palmivora in the Petri dish, C. Gummosis on the stem, D. Healthy stem, no disease symptom.P=P.palmivora, T= Trichoderma, R=Rhizobacteria.
1) Numbers marked by distinct letters differ significantly (P<0.05) according to the LSD test.

Table 3 .
Mean height increment and disease incidence of Soe mandarin inoculated with P. palmivora and treated with Trichoderma spp., Bacillus and Pseudomonas.