The Potential of Rhizophagus intraradices and Trichoderma asperellum to Induce Shallot Resistance against Twisted Disease

Twisted disease caused by Fusarium spp. is one of the primary diseases on shallots with potency to cause enormous losses by causing stunting and bulb rot. One alternative disease control is the induction of plant resistance since the seed stage. The aim of this study was to determine the content of salicylic acid, jasmonic acid, and phenolic compounds of shallot bulb seeds coated with biological control agents as a resistance response to twisted disease. The shallot cultivar used was Crok kuning. The treatments used in this study were the type of biological control agents, including Rhizophagus intraradices and Trichoderma asperellum, combined with and seed coating application time (one month before planting and simultaneously at planting). Biological control agents in the form of powder formulation applied as seed coating material and seeds were stored for one month before planting. The results showed that application of biological agents delayed the occurrence of the twisted disease symptoms. The salicylic acid content from plant treated with R. intraradices at the time of planting was slightly higher than the control. The jasmonic acid content in T. asperellum plants treated at planting was higher than then control. Total phenolic content from plants treated with T. asperellum at planting time was higher than the control. In general, application of biological control agent as seed coat did not result in significant increase in salicylic acid, jasmonic acid nor the phenolic compounds, compared to the pathogen infected control.


INTRODUCTION
Twisted disease is one of the primary diseases of shallot (Allium cepa L. var. ascalonicum). Lestiyani et al. (2016) reported that this disease could be caused by several Fusarium species, namely F. oxysporum F. solani, and F. acutatum. This pathogen can spread through seeds, soil, or water. Symptoms begin with yellowing on the leaf 's tip that then spread to the base, leaves will then wither, twist, and dry up, followed by rotting of the tuber disc and death of the plant (Fitriani et al., 2020). Asaad et al. (2020) reported that this disease could reduce shallot production by more than 50%. Twisted disease is a soil-borne disease where its pathogen can be seed-borne and survive during storage, making this disease even more challenging to control. Disease management among growers primarily rely on the use of synthetic fungicides.
Excessive use of synthetic fungicides has the risk of pathogen resistance and residues both on produce and within the environment. Several preventive measures can be taken to suppress the development of twisted disease, including the application of agricultural lime before planting, the use of resistant varieties, and seed treatment. Seed treatment such as seed coating using synthetic pesticides is a preventive measures commonly implemented by growers. In addition, to synthetic pesticides, alternative coating materials are used including kaolin, ash, limestone, and biological agents.
Some biological control agents have been widely used for seed treatments, including Trichoderma sp. and Arbuscular Mycorrhizal Fungi (AMF). Trichoderma sp. is a biocontrol agent that can activate plant systemic resistance rapidly. Upon root colonization, the plant produces phytohormones such as salicylic acid, Trichoderma asperellum Samuels Lieckfeldt & Nirenberg is a biological control agent that has the potential to control twisted disease. Seed treatment can accelerate root colonization during early stages of plant growth. As a result of root colonization by T. asperellum, there have been reported changes to plant metabolism. A study by Méndez et al. (2020) revealed that T. asperellum BCC1 has potential as a biocontrol agent because it is antagonistic to Sclerotium cepivorum and induces systemic defenses in Allium cepa L. mediated by jasmonic acid and ethylene pathway. Inoculation of T. asperellum can increase total phenolic content in shallots (Ortega-García et al., 2015). Shallot seed coating using T. harzianum was more effective in reducing incidence of damping-off and was able to increase seedling resistance against F. oxysporum and F. solani compared to spraying (Dabire et al., 2016).
Arbuscular mycorrhizal fungi (AMF) is an alternative biological control agents in suppressing soilborne pathogens. In addition to control plant disease, AMF can enhance plant growth and nutrient uptake, especially phosphorus. Plant-AMF interactions can enhance plant defense through changes in secondary metabolic pathways leading to increase tolerance against biotic and abiotic stresses (Kaur & Suseela, 2020). The application of R. irregularis as a coating material for chickpeas (Cicer arietinum L.) has reported to increase plant biomass, nutrient absorption and nutrient concentrations within plants that later have impact on increasing productivity (Rocha et al., 2019). Shallots inoculated with AMF and Trichoderma sp. had low Fusarium wilt disease severity of 0.89% and 1.78%, respectively, at seven weeks after planting. Low disease severity on plants also demonstrated interaction between biocontrol agents and plant host roots (Afiefah et al., 2020). Induced resistance using Rhizophagus irregularis is known to affect the transcription of pathogenesis-related protein (PR) genes thereby increasing systemic acquired resistance (SAR), as well as increasing the regulation of enzymes involved in jasmonic acid biosynthesis in mycorrhiza-treated cotton (Zhang et al., 2018).
The interaction between biological agents and host plants can result in plant defense responses against pathogens (Wardhika et al., 2014). Induction of plant resistance using biological control agents induced systemic resistance by producing various defense compounds. The purpose of this study was to determine the content of salicylic acid, jasmonic acid, and phenolic compounds in shallots coated with biological agents as an indicator of resistance response against twisted disease.

MATERIALS AND METHODS
This research was conducted from August until November 2021 in the Greenhouse and Laboratory of Plant Pathology, Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada. The experiment was performed using a Completely Randomized Design (CRD). The treatment consisted of five treatment that were combination of biological agents (Rhizophagus intraradices N.C. Schenck & G.S. Sm. (synonym Glomus intraradices) and Trichoderma asperellum), pathogen, and application time (one month before planting and simultaneously as planting). The shallot variety used in this research was Crok kuning. The planting medium used was a mixture of soil taken from a shallot plantations in Gotakan Village, Panjatan District, Kulon Progo Regency, mixed with goat manure with a 2:1 ratio. Soil from shallot plantation was used to imitate growing medium from field conditions.

Seed Coating Application with Biocontrol Agents
Rhizophagus intraradices and T. asperellum were mixed with kaolin powder then used as a coating material for the shallot bulbs seed. R. intraradices and T. asperellum were also mixed with kaolin powder and used as the mixture before. The dose of R. intraradices used was 5 g/kg AMF with a spore density of 12.19 spores/g and mixed with 5 g/kg kaolin, while T. asperellum used was 5 g/kg seed with a spore density of 4.6 × 10 6 cfu/g. Seed coating was done by coating the surface of the shallot bulb with the biological agents' mixture until the entire surface of the bulb was covered.
The experiment, was conducted in a completely randomized design (CRD) with five treatments and three replications in each treatment. The combination of treatments for the application of biological agents was: A1C1 = seed coating with R. intraradices at the planting time; A1C2 = seed coating with R. intraradices one month before planting; A1C1= seed coating with T. asperellum at the planting time; A1C2 = seed coating with T. asperellum one month before planting; Control = without seed coating.

Shallot Planting
Shallot planting was done in a 35 × 29 × 12 cm plastic trays, filled with planting media up to 2/3 of the tray. Before planting, approximately 1/3 part of the bottom end of shallot bulbs was first cut and planted using spacing of 10 × 10 cm. Six shallot bulbs were planted per tray.

Pathogen Inoculation
Fusarium solani inoculation was performed 14 days after planting (DAP) by applying 10 mL of spore suspension (1.6×10 6 spores/ml) per plant (Wijoyo et al., 2019). Roots were wounded using a sterile scalpel and spore suspension was then poured onto the roots. Shallot bulbs that were not coated with biological control agents and inoculated with F. solani were used as control. Responses related to induction of plant resistance were observed three weeks after pathogen inoculation, included salicylic acid, jasmonic acid, phenolic content, and calculated Area Under Disease Progress Curve (AUDPC).

Analysis of Salicylic Acid and Jasmonic Acid Content
Analysis of salicylic acid and jasmonic acid content was done at the Laboratory of Agrochemical Residue, Bogor. The analysis was carried out based on methods from Tenhaken and Rubel (1997), which used 1 g of fresh leaves taken from each replicate homogenized with 3 ml of a mixture of methanol and acetone (1:1, v/v), then centrifuged at 5,000 rpm for 10 minutes. The supernatant was discarded, and pellet was extracted using 1 mL of a mixture of methanol and acetone (1:1, v/v) and centrifuged at 5,000 rpm for 10 minutes. The supernatant was then dried using a freeze dryer. The dried residue was suspended using 30% methanol and centrifuged at 5,000 rpm for 10 minutes. The supernatant was analyzed using High-Performance Liquid Chromatography (HPLC). The mobile phase used a solution of methanolsodium acetate buffer 50 mM pH 4.5 (30:70 in 500 mL), homogenized for 10 minutes using a magnetic stirrer with a flow rate of 0.6 mL/minute. Before use, the sample and mobile phase solution was filtered using a 0.45 m RC cellulose acetate filter membrane. The wavelength used was 280 nm with VP-ODS Ultrasphere column type, UV detector at λ 280 nm.

Analysis of Total Phenolic Content
Analysis of total phenolic content was done at the Laboratory of Food Technology and Agricultural Products Analysis, Faculty of Agricultural Technology, Universitas Gadjah Mada. As much as 1 mL of the sample solution was diluted to a total volume of 100 mL, then 1 mL was taken and diluted again to a total volume of 10 mL to obtain total dilution of 1000x (fp = 1000x). The diluted solution of 1 mL was sampled, and then 5 mL of 2% Na 2 CO 3 was added, and left for 10 minutes. Folin-Ciocalteu solution of 0.5 mL was added, then vortexed and left for 30 minutes (Senter et al., 1989;Plumer, 1971 as cited in Susilowati et al., 2014). The absorbance was measured at a wavelength of 750 nm. The phenolic concentration was calculated based on the standard curve obtained from the pure phenol solution of 10-50 ppm as follows:

Determination of Twisted Disease Intensity and Area Under Disease Progress Curve (AUDPC)
Twisted disease intensity was observed once a week and calculated using the following formula (Wibowo et al., 2010): n = number of infected plants having the same score; v = severity score; Z = maximum rating scale number; N = total number of plants observed.
Twisted disease symptom severity scores were categorized as follow: 0 = Symptomless; 1 = Leaf yellowing appears; 2 = The yellowing leaf area developing and leaves began to wilt; 3 = The wilt leaf developing, above half of the leaves yellowed and wilted; 4 = The tuber began to rot; 5 = The plant dies.
The AUDPC value was determined using the formula from (Cooke et al., 2006) as follows: AUDPC = the area under the disease progress curve; n = total number of observations; Yi = assessment of disease intensity at the i th observation; ti = time at the i th observation.

Salicylic Acid (SA) Content in Shallot Leaves
The salicylic acid content from R. intraradices treatment applied simultaneously at planting time slightly increased compared to the control (Figure 1). AMF is considered as a plant pathogen at early stage of initiation, thereby triggering plants to activate defense signals associated with biotrophs pathogens. In the early stages of colonization, AMF triggers salicylic acid production and has strong effect on early stages of AMF formation (Kaur & Suseela, 2020). These results were consistent with research by Poveda et al. (2019), which explained that AMF is biotrophic fungi that make them sensitive to the defense response associated with salicylic acid. This resistance response prevents the fungus from entering the vascular system. During the early interaction between fungi and plants, symbiosis process occurs by suppressing plant defense response associated with salicylic acid, followed by an increase in defense of the jasmonic acid pathway until an appropriate interaction is formed. The alter-nation of salicylic acid to jasmonic acid-dependent defense responses occurred after colonization is established in roots.
In this study, biological control agents were not able to significantly increase the salicylic acid content. This probably have to due to the length of the sampling period, which was conducted at three weeks after pathogen inoculation. According to Yuan et al. (2019), salicylic acid content in cucumber leaf was detected and significantly increased at 96 hours after inoculation of T. longibrachiatum H9.

Jasmonic Acid (JA) Content in Shallot Leaves
The jasmonic acid content in T. asperellum treatment applied simultaneously at planting was relatively higher than control (Figure 2). Trichoderma asperellum is known to mediate induce systemic resistance (ISR) by jasmonic acid signaling pathway. Jasmonic acid is an essential phytohormone in regulating plant resistance system triggered by a beneficial microorganism such as T. asperellum. Pieterse et al. (2014) reported that jasmonic acid regulates the induction of plants' systemic resistance triggered by beneficial microbes such as Pseudomonas, Bacillus, Trichoderma, and AMF. In PGPF, several elicitors with defense activating properties have been identified such as xylanases and cellulases together with proteins and peptides with more specific defense eliciting functions such as Sm1 from T. virens.
Trichoderma asperellum produces hydrophobin class 1 (TasHyd1), which is associated with its attachment to the root surface, protection of hyphal tips against plant defense compounds, and later associated with plant defense responses and induction of plant resistance. In addition, aspartyl protease enzymes identified in T. harzianum and T. asperellum were treated with seed coating using biological agents three weeks after the inoculation of Fusarium solani (A1C1 = seed coating with Rhizophagus intraradices at the same time as planting; A1C2 = seed coating with R. intraradices at one month before planting; A2C1 = seed coating with Trichoderma asperellum at the same time as planting; A2C2 = seedcoating with T. asperellum at one month before planting; Control = without seed coating)  Figure 1. The salicylic acid content from shallot leaves treated with seed coating using biological agents three weeks after the inoculation of Fusarium solani (A1C1 = seed coating with Rhizophagus intraradices at the same time as planting; A1C2 = seed coating with R. intraradices at one month before planting; A2C1 = seed coating with Trichoderma asperellum at the same time as planting; A2C2 =seed coating with T. asperellum at one month before planting; Control = without seed coating) involved in mycoparasitism, increased plant resistance, and induction of secondary metabolites such as phytoalexins (Ent et al., 2009). Nawrocka and Małolepsza (2013) reported that Trichoderma colonization associated with the induction of salicylic acid, jasmonic acid, and ethylene pathways in the same plant, which might indicate the presence of an alternate mechanism of Trichoderma-induced resistance. In addition, it may also imply complicate signaling network connecting SAR and ISR pathways of defense responses, depending on the plant species, Trichoderma strain, and the target pathogen.

Total Phenolic Content in Shallots Leaves
The total phenolic content of each treatment did not show significant differences (Figure 3). The application of biological control agent did not trigger plants to respond pathogen attack by producing higher phenolic compounds that can inhibit pathogen growth.
The colonization phase of biological agents determines the level of phenolic compounds in induced plants. The same results in all treatments were presumably because the biological agents were in the early colonization phase; hence, the phenol accumulation did not show any difference between treatments. Yao et al. (2007) reported that differences in the developmental phase of AMF colonization caused differences in the level of phenolic compounds.
A significant increase occurred in the final phase of colonization when appressoria formation took place in the roots. In the early colonization phase, when arbuscular, vesicles, and spores were formed, the content of phenolic compounds were low.
In this study, the application of T. asperellum did not increase phenolic compound. These results were different from results of Ortega-García et al. (2015) that showed increase of phenolic compounds as a result of the interaction between onion roots and T. asperellum. Such difference in the result was may be due to inadequate root colonization by T. asperellum. Different Trichoderma isolates and onion variety influenced the synthesis of phenolic compounds.

Effect of Biological Agents Coating Application on Twisted Disease Intensity
Symptoms occurred during the first week after pathogen inoculation. The early symptoms observed were leaf yellowing, turning pale green, curling and twisting, then wilting and drying out (Figure 4). The tuber was undeveloped and rot, then the plant dies. This symptom was similar to the twisted disease symptom reported by Lestiyani et al. (2016). Symptoms of wilting and yellowing are presumably secondary symptoms caused by the disruption of the water translocation from the roots to the rest of the plant. A decrease in chloroplasts causes the change in leaf color to yellow due to fusaric acid produced by Fusarium sp., a metabolite compound toxic to plants and not specific to the host only (Agrios, 2005).
Observation of twisted disease intensity on shallot plants treated with biological agent coating showed that the coating could delay the appearance of the twisted disease symptoms ( Figure 5). Symptoms began to appear a week after pathogen inoculation in the control treatment. In plants treated with T. asperellum, symptoms appeared in the second week after pathogen inoculation while in the treatment of AMF, symptoms appeared in the fourth week after pathogen inoculation.
The incubation period of pathogens in AMFtreated plants was longer than in other treatments. This was possibly related to salicylic acid production. In the early stages of colonization, biological agents are recognized as pathogens by plants. The plants respond by activating defense compounds through the salicylic acid pathway results in salicylic acid 61 Artanti et al.: The Potential of Rhizophagus intraradices and Trichoderma asperellum to Induce Shallot Resistance Figure 3. The total phenolic compound concentration in shallot leaves treated with seed coating using biological agents three weeks after the inoculation of Fusarium solani (A1C1 = seed coating with Rhizophagus intraradices at the same time as planting; A1C2 = seed coating with R. intraradices at one month before planting; A2C1 = seed coating with Trichoderma asperellum at the same time as planting; A2C2 = seed coating with T. asperellum at one month before planting; Control = without seed coating)  Zubek et al. (2015) reported increased salicylic acid content and activation of salicylic aciddependent signaling pathways were found in the early stages of development of R. irregularis in roots.
In addition, competition between biological agents and pathogens can prolong the incubation period of pathogens. Competition may occur between the need for nutrients and space for biological agents to colonize roots with Fusarium sp. to infect plants. According to Coronado et al. (2013), root colonization by R. intraradices can protect root tissue from pathogens by competing in same growing space. R. intraradices was able to act as a bioprotectant when >90% roots were colonized. Competition for space with other microbes occurs when R. intraradices colonization in the root system is high. The success of mycorrhizal fungal colonization can be seen in the ability of mycorrhizal fungi to suppress pathogens and delay the appearance of symptoms up to three weeks after pathogen inoculation. These results were consistent with Talanca (2010), who explained that AMF used more carbohydrates before being excreted in the form of root exudates and causes pathogens not to grow. The application of biological agents acted as a bioprotectant that inhibited F. solani infection led to longer incubation period in the treated plants than in control plants. The longer incubation period of the pathogen was probably related to the antagonistic ability of biological agents. Himaya et al. (2021) reported that AMF colonization establishes beneficial interactions for plants and can reduced damage caused by soil borne pathogen and nematodes. These antagonist mechanisms include reducing the site of infection for pathogen through space competition, alteration in root morphology, changes in the composition of root exudate and advancement of crop development, and ultimately plant immune structure stimulation. Marzuki and Assad (2021) reported that the application of Trichoderma sp. at various doses could suppress the development of twisted disease up to 25 days after planting. In addition, Fitriani et al. (2020) also reported that the treatment of a single application of arbuscular mycorrhizal fungi on shallots inoculated with Fusarium oxysporum f.sp. cepae had the most prolonged incubation period compared to other treatments.
Even though the application of biocontrol agents prolonged the incubation period of twisted disease, the treatments could not effectively suppress the disease. The reduction of the disease was only found in the treatment of T. asperellum one month before planting (A2C2) ( Figure 5). Based on the AUDPC value, this treatment was only able to suppress the twisted disease by 12.33% (Figure 6).
Relatively lower AUDPC values were found in the treatment of T. asperellum one month before planting (A2C2) with the value of 327. This treatment was able to suppress the development of F. solani. The ability of Trichoderma to suppress pathogens was probably due to several mechanisms, such as mycoparasitism. According to Zin and Badaluddin (2020), Trichoderma sp. can penetrate host cell walls by forming a hook-shaped structure for penetration. Trichoderma sp. hyphae grow along the host hyphae and secretes cell-wall degrading enzymes and secondary metabolites during the penetration process. This activity could suppress the growth of pathogens and even kill the pathogens. Ismail et al. (2020) reported that shallot treated with the combination of mulch with T. asperellum and compost with T. asperellum showed lower disease incidence than untreated shallot, which were 50%, 43%, and 70%, respectively. T. asperellum penetrated roots and suppress of Fusarium oxysporum f.sp. cepae by direct and indirect mechanism. Direct mechanism is based on the ability of Trichoderma to parasitize and produce antibiotics while indirect mechanisms are based on Trichoderma ability to trigger induce systemic resistance (ISR) and inhibit pathogen. In this study, seed coating treatment using R. intraradices and T. asperellum generally did not result in significant increase of plant defense compounds such as salicylic acid, jasmonic acid and total phenolic content in shallots. However, seed coating treatment using R. intraradices and T. asperellum was able to delay the appearance of twisted disease symptom respectively 4 and 2 weeks after the inoculation of F. solani. 63 Artanti et al.: The Potential of Rhizophagus intraradices and Trichoderma asperellum to Induce Shallot Resistance Figure 5. The development of disease intensity and the time of appearance of twisted disease symptoms on shallot plants coated with biological agents (A1C1 = seed coating with Rhizophagus intraradices at the same time as planting; A1C2 = seed coating with R. intraradices at one month before planting; A2C1 = seed coating with Trichoderma asperellum at the same time as planting; A2C2 = seed coating with T. asperellum at one month before planting; Control = without seed coating) Figure 6. Area Under Disease Progress Curve (AUDPC) value of twisted disease intensity six weeks after the inoculation of Fusarium solani (A1C1 = seed coating with Rhizophagus intraradices at the same time as planting; A1C2 = seed coating with R. intraradices at one month before planting; A2C1 = seed coating with Trichoderma asperellum at the same time as planting; A2C2 = seed coating with T. asperellum at one month before planting; Control = without seed coating) A1C1 A1C2 A2C2 A2C1 Control

CONCLUSION
Seed coating using R. intraradices or T. asperellum did not significantly increase the salicylic and jasmonic acid production compared to the pathogen inoculated control. Application R. intraradices and T. asperellum by seed coating also did not result in significant reduction of twisted disease intensity and AUDPC value, but these biological control agents acted as bioprotectants that delayed the appearance twisted disease symptoms.