Utilization of Arbuscular Mycorrhizal Fungi and Bacillus velezensis Inoculation to Suppressing Twisted Disease of Shallot

Twisted disease is one of the problems in shallot cultivation. The application of the biological agents Bacillus velezensis and arbuscular mycorrhizal fungi is an alternative to overcome disease problems in shallot plantings. The purpose of this study was to determine the effect of the application of B. velezensis and Arbuscular Mycorrhizal Fungi on the growth and health of shallot. The research was conducted in Gotakan, Panjatan, Kulon Progo, and the Phytopathology Laboratory, Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta. Shallot treatments with the application of biological agents were carried out either individually by soaking the bulbs in B. velezensis suspension before planting, coating the bulbs using Arbuscular Mycorrhizal Fungus ( Rhizophagus intraradices ) with a carrier medium of kaolin flour before planting, and a combination of spraying the B. velezensis suspension on plants at two-week intervals during the growth period and coating the bulbs before planting with R. intraradices . The results of the study showed that the shallot-applied single application of B. velezensis reduced the intensity and incidence of twisted disease by 2.51% and 37.6%, respectively. The combination treatment of B. velezensis and R. intraradices was able to increase the resistance of bulbs to post-harvest pathogen Fusarium solani infection, with infected bulbs and areas of 0.70% and 0.71 cm 2 , respectively.


INTRODUCTION
One of the factors causing low production of shallots (Allium cepa L. var.aggregatum) in Indonesia is the presence of Fusarium spp., the pathogen causing twisted disease in shallots.Twisted disease can cause the bulbs to rot, damage roots, cause leaves to grow longer, twist, become pale green but do not wilt, and reduce size and number of marketable bulbs (Nugroho et al., 2015;Prakoso et al., 2017).
Bacillus velezensis is a Plant Growth Promoting Rhizobacteria (PGPR) that produces secondary metabolites with antibiotic properties, which can increase plant resistance and suppress growth and development of pathogens, thereby also having an impact on plant health (Wisanggeni et al., 2023).B. velezensis can also induce plant growth by increasing plants nutrient absorption by, increasing ni-trogen fixation and assisting phosphate dissolution, as well as producing the hormone Indole Acetic Acid (IAA), which can increase plant resistance induction (Sevirasari et al., 2022;Zaid et al., 2022).The use of B. velezensis by soaking and spraying application by Rahma et al. (2020), reported that B. velezensis was able to suppress twisted disease and purple spots development on shallot plants by 67% and increase plant growth and protect shallot bulbs postharvest.Wulan et al. (2022) reported that the use of B. velezensis reduced disease development by 89%, with disease intensity and incidence of 4% and 4%, respectively.
Arbuscular Mycorrhizal Fungi (AMF) are fungi that have a symbiotic relationship with plant roots because they require plant photosynthesis for hyphae growth.AMF hyphae infect plant roots and colonize inside plant roots to help plants absorb nutrients in the rhizosphere zone so that plants can grow and develop more quickly and induce plant resistance to pathogen infections as well as extend the incubation period (Damanik & Suryanto, 2018;Wibowo et al., 2022).There are several species of AMF, namely Acaulospora sp., Glomus etunicatum, Glomus sp., Glomus maniholtis, Gigaspora sp., and Rhizophagus intraradices.The R. intraradices application on shallot plants suppressed growth and development of fusarium wilt disease and increase plant growth compared to the control .This was proven by Fitriani et al. (2019) showing that a single application of R. intraradices resulted in the tallest plants and most number of leaves compared to other endophytic fungi and controls, suppressed pathogen growth with a disease incidence of up to 60% and a latent period of 21 days.Artanti et al. (2022) showed that coating treatment of shallot seeds using R. intraradices suppressed disease symptoms to appear after inoculation using F. solani for four weeks when applications were done either a month before planting or during planting.Palencia et al. (2015) showed that the combination of Glomus intraradices with B. velezensis increased strawberry fruit growth and quality due to the synergistic interaction between B. velezensis colonization on G. intraradices mycelium that formed a biofilm.R. intraradices and B. velezensis interaction as a biological agent on shallots has been done and this this study attempts to explore this matter.Therefore, this research aimed to determine the benefits of using R. intraradices and B. velezensis as biological agents in shallots, either in a single treatment or in combination.

Study Area
The research was conducted between July-December 2021 in Gotakan, Panjatan, Kulon Progo, and the Phytopathology Laboratory, Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta.

Application of Bacillus velezensis and Rhizophagus intraradices on Shallot Bulbs
B. velezensis B-27 was cultured on Yeast Peptone Agar media (5 g yeast, 10 g peptone, and 20 g agar) and incubated at 25 °C for 24−48 hours.The bacteria cultures were then suspended and measured to ob-tain population density of 10 8 cfu/mL for shallot bulb application.Shallot bulbs were soaked in B. velezensis suspension for 30 minutes before planting (Rahma et al., 2020), and the plants were sprayed with B. velezensis suspension during the experiment once every two weeks.R. intraradices with a spore density of 12.03 spores/g obtained previously by filtering zeolite flour containing R. intraradices spores.R. intraradices in zeolite flour and kaolin flour (1:1) were used to coat the bulbs before planting by placing bulbs in mixtures and shaking until bulbs were fully coated.
The seeds used were the 'Siyem' local variety obtained from farmers.This research was arranged as a Randomized Complete Block Design (RCBD) with four treatments, three blocks, and three replications.The treatments consisted of Bv (B.velezensis soaking application), Ri (R. intraradices coating application), Bv+Ri (combination of R. intraradices coating treatment before planting and spraying B. velezensis at once every two weeks), and control (without application of B. velezensis & R. intraradices).Treated seeds were then planted in ± 100 cm × 200 cm beds with distances of 15 cm × 20 cm, and NPK and ZA fertilizers were applied at a dose of 15−200 kg/500 m 2 and 25 kg/500 m 2 , respectively.

Disease Incidence, Disease Intensity, and Area Under Disease Progress Curve (AUDPC) Analysis
Disease incidence and intensity were measured every two weeks.Disease incidence was calculated based on the number of diseased plants at each observation, and the percentage was calculated using the formula according to Ismiyatuningsih et al. (2016): The intensity of twisted disease was carried out using a scoring method for symptoms on shallot plants (Table 1) and calculated using the formula according to Widyaningsih et al. (2019)

Data Analysis
The data obtained were analyzed using ANOVA and continued with the Tukey Test with a level of 5%.

Disease Incidence, Disease Intensity, and Area Under Disease Progress Curve (AUDPC) Analysis
Highest disease incidence occurred in the control treatment (65.6%), and lowest was in the B. velezensis treatment (37.6%).R. intraradices and combination treatments resulted in 44.3% and 41%, respectively.Disease incidence were significantly different in the biological agent treatment compared to the control treatment.Likewise, twisted disease intensity showed significantly different results in the biological agent treatment compared to the control treatment, with the highest disease intensity in the control treatment (7.28%) and the lowest in the B. velezensis treatment (2.51%).Meanwhile, the disease intensity value in R. intraradices and combination treatment were 2.78% and 2.67%, respectively (Table 2).This research showed that B. velezensis and R. intraradices, either applied individually or combined, were able to suppress the growth and development of twisted disease on shallots.B. velezensis ability to inhibit pathogen growth was reported by Moradi-Pour et al. (2021), mentioning that the presence of siderophore compounds affected the utilization of Fe that can inhibit Rhizoctonia solani growth.
Research by Jiang et al. (2019) showed that B. velezensis F21 had antagonistic activity against pathogenic fungi with hydrolase activity such as protease, ferric enzyme, glucanase, and cellulose, but did not show chitinase activity.

Postharvest Shallot Resistance
Postharvest shallot resistance was tested using bulbs from previous field research.Fusarium solani fungus isolates for inoculation were obtained from the Plant Disease Science Laboratory collection, which were cultured on Potato Dextrose Agar medium (200 g Potato extract, 20 g dextrose, and 15 g agar).Before inoculated, the bulbs were washed with running water and dried, then disinfected with 70% ethanol for 2 minutes and rinsed using sterile water for 1 minute.After that, the bulbs were inoculated by cutting the F. solani inoculum using a cork drill with a diameter of 0.8 cm.Pieces of Fusarium solani isolates mycelium were attached to the bulb disc that had been injured by piercing using a sterile preparation needle.The inoculated bulbs were then placed in a closed container with sterile cotton wool and sterilized water then incubated for seven days at room temperature.
On the 7 th day after inoculation, the bulbs were cut open, and the area of necrosis symptoms were measured using a millimeter block and calculated using the following formula: Bacillus velezensis and R. intraradices effectiveness in suppressing disease can be determined by calculating the Area Under Disease Progress Curve (AUDPC).The AUDPC value for the control treatment had the highest value (18.28%.days), while the lowest was in the B. velezensis treatment (5.16%.days).The combination treatment had an AUDPC value of 5.17%.days and the R. intraradices treatment of 6.22%.days(Table 2).Smaller AUDPC values indicate better control (Widyaningsih et al., 2017).Therefore, B. velezensis, R. intraradices, and combination treatments were able to suppress the twisted disease growth in shallot plants.
Bacillus velezensis can produce several secondary metabolites with potential biocontrol activity, namely antibiotic properties by inducing antibacterial metabolites and lipopeptides that activate plant resistance in the presence of surfactin toxins and lipopeptin and later triggering plant growth hormones regarding to nutrient absorption and competition against Bacillus with pathogens ( Ye et al., 2018).Secondary metabolites (fengycin, iturin, and surfactin) produced by B. velezensis are Bacillus cyclic lipopeptides (CLPs) or antifungal cycles, which contribute to fungal phytopathogenic activity to penetrate cell membrane, forming ion channels in the cell membrane pores, causing osmotic pressure imbalance in the membrane, damaging membrane and destroying pathogenic cells (Chen et al., 2018).Secondary metabolites of B. velezensis can damage fungal cell walls and cell membranes by releasing proteases, for example extracellular protease (aprE), bacillus peptidase (bpr), and β-glucanase that can damage fungal mycelium (Wang et al., 2020).

Resistance of Shallot Bulbs to Fusarium solani Infection in the Postharvest Period
Results showed the percentage of infected bulbs in treated with only B. velezensis (0.77) and with combination treatment (0.70) were significantly different from the control treatment (1.12).However, the single treatment R. intraradices (0.84) was not significantly different compared to all treatments.Meanwhile, the extent of bulb infection in the single treatment of B. velezensis, R. intraradices or the combination of both were not significantly different compared to the control (Table 3).These results indicate that the combination treatment of B. velezensis and R. intraradices can increase the resistance of post-harvest bulbs to F. solani infection.
R. intraradices root infection will expand the reach of the roots due to the external hyphae produced from mycorrhizal roots.R. intraradices infection is also able to increase the availability and supply of phosphate to plants, and around 80% of phosphorus requirements in plants are supplied by R. intraradices (Nadeem et al., 2014).Jeffries et al. (2003) reported mutualistic interaction between PGPR and mycorrhizal fungi.PGPR will stimulate the growth of mycorrhizal fungal hyphae by increasing cell permeability, making it easier for mycorrhizal fungal to infect roots.Meanwhile, R. intraradices will increase the activity of nitrogen-fixing and phosphate-solubilizing bacteria.Phosphate absorbed by plants plays a role in structuring and stabilizing cell walls by strengthening and increasing cell density so that cell walls are not easily damaged (Wibawa et al., 2023).The hardness of shallot bulbs can be influenced by the change in water-insoluble peptin (protopectin) into water-soluble pectin (Wibawa et al., 2023).Changes in these components can help maintain the durability of the bulbs.Besides, the ability of bacteria and fungi to produce hormones and enzymes, which cause the epidermal cells of shallot bulbs to become strong and thick, can resist and make it difficult for pathogens to penetrate directly.Research conducted by Ilmiah et al. (2021) showed that the application of B. velezensis with goat manure could increase the content of total phenolic compounds and antioxidant compounds in snake fruit.In addition, Al-Askar and Rashad (2010) reported that the application of R. intraradices was able to stimulate the production of plant resistancerelated enzymes, such as polyphenol oxidase and peroxidase.

CONCLUSION
Based on this research, B. velezensis and R. intraradices application either individually or combined to shallot was able to suppress the growth and development of twisted disease.The combination application of B. velezensis with R. intraradices was also able to increase postharvest bulb resistance.
as follows: Information: n = number of diseased plants per category; v = score value per category; N = number of plants observed; Z = the highest score value.Incidence of disease = Number of diseased plant × 100% Total number of plants observed Intensity of disease = Σ(n × v) × 100% N × Z Analysis of the Area Under Disease Progress Curve (AUDPC) value was calculated based on the graph of disease intensity development using the formula according to Rahma et al. (2020) as follows: Remarks: Y i+1 = Observation data i + 1; Y i = Observation data i; t i+1 = Observations time i +1; t i = Observation time i; n = Total plants ISSN 1410-1637 (print),ISSN 2548-4788 (online) B. velezensis was able to suppress the development of Fusarium wilt disease in watermelon plants by 80.35% in field settings and 65.81% in greenhouse settings.In addition, B. velezensis F21 was able to induce resistance in watermelon plants affected by fusarium wilt disease due Utilization of Arbuscular Mycorrhizal Fungi and Bacillus velezensis Inoculation (Köycü & Oezer, 1997)), ISSN 2548-4788 (online) 105Abdullah et al.:Source:(Köycü & Oezer, 1997)

Table 2 .
Effects of Bacillus velezensis and Rhizophagus intraradices application on the development of incidence, intensity and AUDPC of twisted disease on shallot plants *Remarks: Values followed by the same letters in the same column are not significantly different.Description: Bv (B.velezensis soaking application), Ri (R. intraradices coating application), Bv+Ri (combination of R. intraradices coating treatment before planting & spraying B. velezensis once every two weeks ), and control (without application of B. velezensis & R. intraradices).