J. Trop.

Pratiwi etPlant
al. Pests Dis. ISSN:spp.
Response of five shallot varieties applied with Bacillus 17
1411-7525
Vol. 24, No. 1, March 2024 E-ISSN: 2461-0399
Pages: 17–27 DOI : 10.23960/j.hptt.12417-27

RESEARCH PAPER

Response of five shallot varieties applied with Bacillus spp. against twisted disease

Annisa Hasta Pratiwi, Arif Wibowo, Tri Joko, Ani Widiastuti, & Siti Subandiyah

Manuscript received: 1 August 2023. Revision accepted: 8 September 2023. Available online: 8 January 2024.

ABSTRACT

The use of several shallot varieties applied with the biological agent Bacillus spp. is one of the most developed methods
of controlling twisted disease, as it is safe and efficient. The large number of shallot varieties released to farmers requires
the selection of varieties with the best resistance response to twisted disease. This study aimed to determine the different
responses between five local shallot varieties treated with a combination of Bacillus velezensis B-27 and B. cereus RC76
against the twisted disease. This study was conducted in a greenhouse and on the field using Tajuk, Bima Brebes, Bauji,
Crok Kuning, and Manjung varieties, which were dipped and sprayed with a B. velezensis B-27 and B. cereus RC76. The
treatment of B. velezensis B-27 and B. cereus RC76 on five varieties showed a good response to suppressing twisted disease.
The twisted disease incubation period in five varieties treated with the combination of B. velezensis B-27 and B. cereus RC76
showed a slower result than the control, the disease incidence and intensity could be reduced by 70–90%. The best resistance
response of varieties treated with the combination of B. velezensis B-27 and B. cereus RC76 was shown by Tajuk compared
to the other four varieties.
Key words: Bacillus cereus RC76, Bacillus velezensis B-27, Fusarium disease, Tajuk variety

INTRODUCTION bulbs in storage, accounting for around 30–40% of losses

(Le et al., 2021).
Twisted disease is one of the crucial diseases in In the cultivation of shallots, the use of resistant
shallots, which causes a decrease in shallot production. varieties is a common practice for controlling twisted
It is caused by the infection of the pathogenic fungus disease. Planting shallots with resistant varieties is
Fusarium spp., which can result in abnormal plant chosen for its efficiency, simplicity, and environmental
growth, leaf chlorosis, bulb rot, and plant death friendliness. Local varieties like Tiron and Bauji have
(Emeliawati et al., 2022). An early symptom of twisted been reported independently to exhibit high resistance to
disease is the curling of pale green and yellowish leaves twisted disease (Wiyatiningsih et al., 2009; Wibowo et
from the base, along with stunted plant growth. This al., 2016). However, a current challenge is the extensive
symptom can progress to rotting of the bulbs and make number of varieties available to farmers, necessitating
the plants easily uprooted (Rohma & Wahyuni, 2022). further studies to identify several varieties that are
The three species of Fusarium that cause resistant to twisted disease.
different symptoms of twisted disease are F. solani, F. The utilization of bacterial biological agents is
oxysporum, and F. acutatum. F. solani and F. acutatum also carried out as an effort to control twisted disease,
induce symptoms such as yellowing, dryness, twisting, alongside the use of resistant varieties. The use of
and wilting of leaves in plants. Moreover, F. solani, F. bacterial biological agents for controlling plant diseases
oxysporum, and F. acutatum cause rotting symptoms has gained widespread development and application.
in shallot bulbs (Lestiyani et al., 2021). Fusarium spp. Bacillus spp. is a type of bacteria extensively employed
infect shallot plants during the growing season and cause due to its role as Plant Growth Promoting Rhizobacteria
yield loss of up to 50% in the field (Wiyatiningsih, 2021). which stimulates plant growth and can suppress plant
Additionally, the inoculum of Fusarium spp. can survive diseases (Khan et al., 2017). Bacillus spp. can enhance
in the soil or carried by bulbs and causing damage to plant resistance to disease either directly or indirectly by
competing for space or releasing secondary metabolites
Corresponding author: that are antifungal and supportive of plant growth
Arif Wibowo (arif_wibowo@ugm.ac.id)
(Ntushelo et al., 2019; Sudana & Lotrini, 2005).
Department of Plant Protection, Faculty of Agriculture, Bacillus velezensis B-27 and Bacillus cereus
Universitas Gadjah Mada, Jln. Flora No. 1, Bulaksumur, RC76, employed in this study, had been tested and
Sleman, Yogyakarta, 55281 Indonesia. demonstrated their potential as bacterial biological
18 J. Trop. Plant Pests Dis. Vol. 24, No. 1, 2024: 17–27

control agents. In a previous study, it was reported that planted varieties in shallot cultivation. The selection was
B. velezensis B-27, using the bulb-dipping technique, also informed by literature that states discusses varieties
could reduce twisted disease and purple blotch by 67% in terms of production, weather adaptation, and resistance
in shallots of Crok Kuning variety (Rahma et al., 2020). to plant diseases. The Tajuk variety demonstrated has
Bulbs dipping using a combination of B. velezensis good adaptation to seasons (Maharijaya et al., 2016) and
B-27 and B. cereus RC76 could suppress twisted disease is widely cultivated on the island of Java. About 71.43%
by 84% and increase the induction of plant resistance of Central Java farmers have been planting the Bima
to twisted disease in Bima Brebes variety (Wulan et Brebes variety since its release, according to Pusdatin
al., 2022). B. velezensis B-27 was also reported to (2013). Crok Kuning is a local variety from Bantul, the
increase antioxidant activity, total phenol, and flavonoid location of the research experimental fields, and has a
compounds, which play a role in metabolic pathways high yield potential. The Bauji and Manjung varieties
and plant resistance to disease (Ilmiah et al., 2021; hold superior variety status according to information
Djaenuddin et al., 2018). from the Ministry of Agriculture.
Research by Rahma et al. (2020) and Wulan et al.
(2022) applied B. velezensis B-27 and B. cereus RC76 Experimental Design. The greenhouse experiment
to shallots using only one variety and/or one application used a completely randomized design with two factors
technique. Research regarding the application of a and three replications. The first factor was five shallot
combination of B. velezensis B-27 and B. cereus RC76 varieties, and the second factor was the treatment, which
through bulb dipping and plant spraying on five varieties, consisted of:
namely Tajuk, Bima Brebes, Bauji, Crok Kuning, and A. (Bacillus spp.+ inoculation): Dipping the bulbs before
Manjung, against twisted disease had not been carried planting and spraying shallot plants using a combination
out before. Therefore, in this study, five local shallot of B. velezensis B-27 and B. cereus RC76 + inoculation
varieties were tested, treated by bulbs dipping and plants of Fusarium acutatum pathogen.
spraying with a combination of B. velezensis B-27 and B. B. (Inoculation): Inoculation of F. acutatum and without
cereus RC76. This study aimed to determine the increase the application of a combination of B. velezensis B-27
in resistance response of the five shallot varieties treated and B. cereus RC76.
with a combination of B. velezensis B-27 and B. cereus C. (Non-inoculation): Without inoculation of F. acutatum
RC76 to twisted disease. and without the application of a combination of B.
velezensis B-27 and B. cereus RC76.
MATERIALS AND METHODS The experimental field used a randomized block design,
where the treatment involved five shallot varieties treated
Research Site. The research was conducted in two by bulb dipping before planting and plant spraying using
locations: in the greenhouse of the Department of Plant a combination of B. velezensis B-27 and B. cereus RC76.
Protection, Faculty of Agriculture, Universitas Gadjah
Mada, and in Samas Beach Sand Field, Sanden District, Propagation of Bacillus spp. B. velezensis B-27 and
Bantul Regency, D.I. Yogyakarta. The sandy field is of B. cereus RC76 were two bacterial species used in this
coastal sandy land with regosol eutric soil type and a sand study as biological control agents for twisted disease.
dunes landscape type. These two experiments used five The two bacterial isolates were obtained from the
local shallot varieties that are frequently cultivated by collection of the Laboratory of Plant Diseases, Faculty of
farmers in Java, namely Tajuk, Bima Brebes, Bauji, Crok Agriculture, UGM. They were cultured in Yeast Peptone
Kuning, and Manjung (description in Table 1). Agar medium (0.5% yeast, 1% peptone, and 1.5% agar)
The selection of five varieties in this study was for 48 hours. After that, the isolates were dissolved in
based on local farmers’s habits regarding the most sterile distilled water to obtain the required bacterial

Table 1. Description of five shallot varieties were used

No. Varieties Origin Variety character
1. Tajuk Nganjuk, East Java Well adapted at dry and wet season
2. Bima Brebes Brebes, Central Java Moderately resistant to bulb rot disease
3. Bauji Nganjuk, East Java Tolerant to Fusarium sp. and Spodoptera sp.
4. Crok Kuning Bantul, Yogyakarta High production and well adapted in lowland
5. Manjung Pamekasan, East Java Well adapted in lowland, dry and wet season
Pratiwi et al. Response of five shallot varieties applied with Bacillus spp. 19

suspension with a density of 108 CFU/mL (Rahma et al., Treatment of Bacillus spp. on Five Varieties in the
2020). The suspension of B. velezensis B-27 was mixed Field. Field research was conducted during the dry
with a suspension of B. cereus RC76 in a ratio of 1:1 as season from the end of March until May. Shallot planting
a combined suspension before being applied to shallots. in the field was carried out in 1 × 10 m beds with three
replicate blocks. The total number of beds used were 15,
Treatment of Bacillus spp. in the Greenhouse. Prior and the total number of shallot plants planted in one bed
to inoculation and planting, the bulbs of five shallot was 300. The bulbs of five shallot varieties were dipped
varieties (treatment A) were dipped in a combined in a bacterial combined suspension of B. velezensis B-27
suspension of B. velezensis B-27 and B. cereus RC76 and B. cereus RC76 at a density of 108 CFU/mL with a
at a density of 108 CFU/mL, which had previously been ratio of 1:1. Each variety was dipped in 300 mL of the
obtained, using 10 mL per bulb for 30 min. Then, the bacterial combination added to 3000 mL of water for 30
bulbs were air-dried (Wulan et al., 2022). Shallot bulbs min, then air-dried (Wulan et al., 2022). The five shallot
with treatments B and C were dipped in sterile water varieties were then planted in beds with a spacing of 20
and then air-dried. × 15 cm.
The next treatment involved spraying the shallot One week after planting, the shallot plants were
plants in treatment A with a combination of B. velezensis first sprayed with a combination of B. velezensis B-27 and
B-27 and B. cereus RC76 every two weeks. A combined B. cereus RC76 at a density of 108 CFU/mL. Spraying
suspension of B. velezensis B-27 and B. cereus RC76 at a shallots in the field was done using a 16 L automatic
density of 108 CFU/mL were applied to shallot plants in knapsack sprayer. The combination of B. velezensis
treatment A using a 1000 mL hand sprayer. Each shallot B-27 and B. cereus RC76 was applied to each bed with
plant was sprayed with 10 mL suspension every two 300 mL of bacterial suspension added to 3000 mL of
weeks starting from seven days after planting (DAP). water, or about 10 mL per plant. The time interval for
The time interval for spraying plants were at 7, 21, and spraying the plants was once every two weeks, recorded
35 DAP, conducted around 7–8 AM in the morning. at 7, 21, and 35 DAP, which was done around 7–8 AM
in the morning.
Inoculation of Pathogenic Fungi in the Greenhouse.
The inoculation of pathogen was carried out during Maintenance of Five Shallot Varieties in the Filed.
the shallot planting process using F. acutatum isolates Maintenance for shallot was carried out by watering
from the collection of the Laboratory of Plant Diseases, plants every morning and evening automatically using
Faculty of Agriculture UGM. F. acutatum was cultured an irrigation hose spraying method. Furthermore, the
in Potato Dextrose Agar (PDA) medium, which consisted plants were fertilized using SP36 at a rate of 150 kg/ha
of a broth of 250 g potatoes, 20 g dextrose, and 20 g agar at 7 DAP, 150 kg/ha of urea, 200 kg/ha of ZA and 150
dissolved in 1000 mL of distilled water and sterilized kg/ha of KCl at 2, 4 and 6 weeks after planting. Pest
in an autoclave at 120 ºC. The F. acutatum isolate from control was also implemented by applying chemical
laboratory collection was plugged using a cork borer insecticides with the active ingredient Sipermethrin
with a diameter of 1 cm, and then the mycelium pieces and biological insecticides with the entomopathogenic
were transferred into a new PDA medium to propagate Beauveria bassiana to control armyworm pests and
F. acutatum. F. acutatum was incubated for seven days thrips on shallot plants every week.
at a room temperature of 22–25 ºC. The fungal isolate
was then dissolved in sterile distilled water to obtain a Observation of Incubation Period, Incidence,
suspension of F. acutatum conidia at a density of 1 x 106 Intensity of Twisted Disease and Area Under Disease
conidia/mL (Lestiyani et al., 2021). Progress Curve (AUDPC). The incubation period
Three bulbs were planted in each polybag of twisted disease was observed in the greenhouse
measuring 35 × 35 cm, consisting of sterile soil with experiment every day from the inoculation of F. acutatum
three replicate polybags per treatment. The total until the onset of twisted disease symptoms in shallot
number of polybags used was 45, and the bulbs used plants (Prakoso et al., 2016). Symptoms of twisted
were 135 shallot bulbs. The conidial suspension of F. disease observed in shallots included dry, yellowing, or
acutatum, which had previously been obtained, was then twisting leaves, and rotting bulbs.
inoculated by pouring the conidial suspension into bulbs The incidence of twisted disease was observed
of five shallot varieties in treatments A (Bacillus spp. + in the greenhouse and in the field every week after the
inoculation) and B (inoculation) each with 10 mL per application of the combination of B. velezensis B-27 and
shallot bulb (Wijoyo et al., 2020). B. cereus RC76, at 14, 28 and 42 DAP. The incidence of
20 J. Trop. Plant Pests Dis. Vol. 24, No. 1, 2024: 17–27

twisted disease was determined by calculating the total Moderately resistant (10–30%); Susceptible (30–50%);
number of diseased plants in each polybag or treatment Very susceptible (50–100%).
bed and then using the formula (Wiyatiningsih et al., Furthermore, with an observation interval of 14
2009): days, the disease intensity is used to calculate AUDPC
n using the formula (Simko & Piepho, 2012):
DI = N # 100%

AUDPC = / i (
n - 1 Yi + Yi + 1
DI = Disease incidence; 2 ) (ti + 1 - ti)
n = Number of diseased plants observed;
N = Number of plant observed. Yi+1 = Observational data-i + 1;
Yi = Observational data-i;
The intensity of twisted disease was observed in all ti + 1 = Observational time-i + 1;
shallot plants in each treatment in polybags and beds, ti = Observational time-i.
and then categorized based on symptom scoring and
calculated using the formula (Hadiwiyono et al., 2020): Statistical Analysis. Data were analyzed using analysis
/ (n # v) # 100% of variance and further tested using Duncan Multiple Test
DS = Range (DMRT) with a 95% confidence level. Analysis
Z#N
was performed using IBM SPSS Statistics software
DS = Disease severity; version 26.
Z = Highest score;
N = Total plants observed; RESULTS AND DISCUSSION
n = Number of infected plants with a certain
Incubation Period of Twisted Disease. The incubation
score;
period of twisted disease in five varieties treated with
v = Disease severity score. the combination of B. velezensis B-27 and B. cereus
RC76 showed a slower onset of disease symptoms
Scoring is based on Rahma et al. (2020) with modifications: compared to the inoculation treatment. Symptoms of
a score of 0 = no symptoms, 1= 1–20% leaves turning twisted disease in five shallot varieties treated with the
yellow and twisting, 2= 21–40% leaves turning yellow, combination of B. velezensis B-27 and B. cereus RC76
dry and twisting, 3= 41–60% leaves turning yellow, dry appeared the fastest in the Crok Kuning variety at 21 days
and twisting, 4= 61–80% leaves turn yellow, dry, twisting after inoculation (DAI), while the slowest was shown
and the bulbs rot, 5= dead plants). in the Tajuk and Manjung varieties at 28 DAI. In the
Disease resistance criteria are determined based inoculation treatment, the varieties showing the fastest
on criteria that have been modified by Herlina et al. incubation period were the Bima Brebes and Manjung
(2021). The categories for resistance criteria are based varieties at 13 DAI, and the slowest at 21 DAI for the
on the percentage of twisted disease intensity in shallots, Tajuk variety (Figure 1). Study by Cahyaningrum et al.
including: Very resistant (0%); Resistant (1–10%); (2020) showed that shallots inoculated by Fusarium sp.
Incubation period (day (s) after inoculation)

35
Bacillus spp.+ inoculation Inoculation
30 28 28
25 23 24
21 21
20 18
15 13 14 13
10
5
0
Tajuk Bima Brebes Bauji Crok Kuning Manjung
Figure 1. The incubation period of twisted disease on five shallot varieties after applying the combination of
Bacillus velezensis B-27 and Bacillus cereus RC76.
Pratiwi et al. Response of five shallot varieties applied with Bacillus spp. 21

exhibited twisted disease symptoms in 7–12 DAI. The can synthesize surfactin, bacillomycin-D, fengycin,
study by Prakoso et al. (2016) demonstrated the twisted siderophore, difficidin, and several other secondary
disease incubation period for Bauji Magetan variety was metabolite compounds. Fengycin can inhibit mycelium
at 14 days, for Manjung at 17 days, and Bauji Nganjuk growth, change the morphology of fungal hyphae and
showed the longest period at 20 days. The incubation conidia cell walls, and inhibit fungal spore germination
period for twisted disease in Crok Kuning variety is (Rabbee et al., 2019). B. cereus is capable of producing
reported at 11 DAI (Wibowo et al., 2016). secondary metabolites and organic components that
Shallots inoculated by F. acutatum showed are antifungal and increase plant growth, for example,
different symptoms of twisted disease in the five shallot indole acetic acid, ACC deaminase, siderophores and
varieties tested. Tajuk and Bauji varieties in the combined phosphates (Zhou et al., 2021).
treatment of B. velezensis B-27 and B. cereus RC76, as
well as the inoculation treatment, had the same slow Incidence and Intensity of Twisted Disease. The
incubation period compared to the other four varieties. twisted disease incidence and intensity were presented in
This result indicated that Tajuk and Bauji may have the form of a percentage, which showed the magnitude of
potential resistance to twisted disease from the start, and the disease incidence caused by pathogens quantitatively.
then the resistance increased after being treated with a The results of the study in the greenhouse showed that
combination of B. velezensis B-27 and B. cereus RC76. there was no interaction between treatments and varieties
The difference in the incubation period was due to the on the incidence and intensity of twisted disease. The
resistance response factors of different shallot varieties treatment of B. velezensis B-27 and B. cereus RC76
to twisted disease. The emergence of twisted disease was significantly different compared to the inoculation
symptoms in all varieties of the inoculation treatment treatment in terms of twised disease incidence and
was in the range of 13–21 DAI, which appeared faster intensity. Five shallot varieties also showed significant
than in the combined treatment of B. velezensis B-27 differences between varieties to twisted disease incidence
and B. cereus RC76. and no significant difference in twisted disease intensity.
Bima Brebes variety showed the fastest incubation Overall, the percentage of the disease incidence in Tajuk
period in the inoculation treatment and the second fastest variety showed the smallest value of 51.85%, which
in the combined treatment of B. velezensis B-27 and B. was significantly different from Bima Brebes, 70.37%.
cereus RC76. The range of the symptoms appearance Bauji, Crok Kuning and Manjung varieties showed
in Bima Brebes was similar to Crok Kuning, which no significant difference with the disease incidence of
showed the fastest incubation period in the combined 66.67%; 62.96% and 66.67%, respectively (Table 2).
treatment of B. velezensis B-27 and B. cereus RC76 and The combined treatment of B. velezensis B-27 and
the second fastest in the inoculation treatment. Based on B. cereus RC76 in the greenhouse showed a significant
the time span of symptoms appearance, the Manjung effect compared to inoculation treatment on the disease
variety showed the best response, with the greatest intensity with a value of 26.67%. The result indicated that
period ranged between treatment by Bacillus and non- the treatment of B. velezensis B-27 and B. cereus RC76 on
Bacillus, namely at 28 DAI and 13 DAI. A comparison five shallot varieties in the greenhouse could reduce the
of the distance between the disease incubation period in disease intensity by up to 73.33%. Among the five shallot
the varieties treated with bacteria and the non-bacterial varieties tested, disease intensity results did not show
treated varieties showed the potential of a combination a significant difference in the five varieties tested. The
of B. velezensis B-27 and B. cereus RC76. lowest disease intensity was shown by Tajuk and Bauji
The role of the treatment with B. velezensis and B. varieties but was not significantly different from Bima
cereus is also one of the important factors in the period Brebes, Crok Kuning and Manjung. The suppression of
of the emergence of twisted disease symptoms. The twisted disease based on the disease intensity of five
fastest incubation period in the combined treatment of B. shallot varieties in all treatments ranged from 71.85%
velezensis B-27 and B. cereus RC76 was the same as the to 77.78% (Table 3). The ability of B. velezensis B-27
slowest incubation period in the inoculation treatment. and B. cereus RC76 in combination to suppress twisted
This showed that the combined treatment of B. velezensis disease in the greenhouse, based on the study by Wulan
B-27 and B. cereus RC76 played a role in slowing the et al. (2022) showed that the twisted disease intensity
infection process of F. acutatum and the emergence of was reduced by up to 90% with the bulb-dipping method.
twisted disease symptoms. Several studies have shown The field experiment was carried out by applying
that Bacillus spp. have the potential to inhibit pathogens a combination of B. velezensis B-27 and B. cereus RC76
by producing secondary metabolites. B. velezensis in five varieties to compare the resistance response
22 J. Trop. Plant Pests Dis. Vol. 24, No. 1, 2024: 17–27

Table 2. The incidence of twisted disease on five shallot varieties applied with the combination of Bacillus
velezensis B-27 and Bacillus cereus RC76 in the greenhouse
Varieties
Treatments Average*
Tajuk Bima Brebes Bauji Crok Kuning Manjung
Bacillus spp. + inoculation 55.56 88.89 88.89 77.78 88.89 80.00 b
Inoculation 100.00 100.00 100.00 100.00 100.00 100.00 a
Non-inoculation 0.00 22.22 11.11 11.11 11.11 11.11 c
Average* 51.85 b 70.37 a 66.67 ab 62.96 ab 66.67 ab (-)
The sign (-) indicates that there is no interaction between treatments and varieties. *= Values followed by different
letters in the same treatment and column showed significant differences (DMRT; α= 0.05).
Table 3. The intensity of twisted disease on five shallot varieties in the greenhouse
Varieties
Treatments Average*
Tajuk Bima Brebes Bauji Crok Kuning Manjung
Bacillus spp. + inoculation 13.33 40.00 22.22 33.33 24.45 26.67 b
Inoculation 53.33 40.00 42.22 37.78 42.22 43.11 a
Non-inoculation 0.00 4.45 2.22 2.22 2.22 2.22 c
Average ns
22.22 28.15 22.22 24.44 22.96 (-)
The sign (-) indicates that there is no interaction between treatments and varieties. *= Values followed by different
letters in the same treatment and column showed significant differences (DMRT; α = 0.05). ns= non significantly
different (ANOVA; α = 0.05).
between varieties against twisted disease incidence and each variety can be caused by differences of the variety,
intensity. The twisted disease incidence and intensity in treatments and the environment used for the research.
the field showed significantly different results among The treatment of a combination of B. velezensis
the five shallot varieties. Observations made from 14 B-27 and B. cereus RC76 affected the twisted disease
to 42 DAP showed a different progression of disease incidence and intensity. Overall, the combined treatment
incidence. At the 42nd day of observation, the lowest of B. velezensis B-27 and B. cereus RC76 on the Tajuk
disease incidence in the field was shown by Tajuk of variety had the lowest disease intensity, namely 13.33%
16.57%, which was significantly different from Bima in the greenhouse and 6.32% in the field. The low
Brebes, Bauji, Crok Kuning and Manjung. The result incidence and intensity were influenced by the ability
of the disease intensity showed that the percentage was of B. velezensis B-27 and B. cereus RC76 to suppress
directly proportional to the disease incidence, with the twisted disease directly and indirectly. Wulan et al.
lowest disease intensity shown by Tajuk at 6.32%, which (2022) stated that shallots dipped in B. cereus RC76 and
was significantly different from Bima Brebes, Bauji, Crok B. velezensis B-27 had an intensity of twisted disease of
Kuning and Manjung (Table 4). 10%, resulting in a 72.2% suppression of twisted disease.
Observations made from 14 to 42 DAP showed In the study by Rahma et al. (2020), B. velezensis B-27
an increase and differences between the five varieties could inhibit twisted disease in shallots by the bulb-
in disease intensity. The highest development of dipping technique, where the intensity of twisted disease
twisted disease was shown by the Bauji variety, and was shown to be the lowest, at 0.15%.
the lowest was shown by Tajuk variety. The difference In previous studies, it has been known that
in disease intensity among the five shallot varieties Bacillus spp. could produce secondary metabolites that
resulted in variations in the resistance levels of the can cause a low intensity of disease due to pathogens
different varieties. Based on the disease intensity of field (Wulan et al., 2022). Compounds produced by Bacillus
experiment, five varieties were classified into resistance spp. play a role in inhibiting the growth of pathogens,
criteria according to Herlina et al. (2021) with slight assisting the development of bacteria, and inducing
modifications. The results showed that Tajuk and Bima plant resistance to disease. B. cereus is able to produce
Brebes were classified as resistant, while Bauji, Crok hydrolytic enzymes and volatile components that can
Kuning and Manjung were classified as moderately damage cell walls and inhibit the growth of the mycelium
resistant (Table 5). Differences in resistance responses by of Fusarium oxysporum (Ramírez et al., 2022). B.
Pratiwi et al. Response of five shallot varieties applied with Bacillus spp. 23

Table 4. The twisted disease incidence and intensity on five shallot varieties applied with the combination of
Bacillus velezensis B-27 and Bacillus cereus RC76 in sandy field
Disease incidence (%) Disease intensity (%)
Varieties
14 DAP* 28 DAP ns
42 DAP* 14 DAP* 28 DAPns 42 DAP*
Tajuk 8.87 ab 9.45 16.57 b 1.85 ab 3.20 6.32 c
Bima Brebes 8.93 ab 12.35 22.59 ab 1.90 ab 4.12 9.43 b
Bauji 6.70 b 13.57 25.82 ab 1.38 b 5.17 12.71 a
Crok Kuning 12.18 a 12.73 28.19 a 2.53 a 4.34 11.21 ab
Manjung 9.97 ab 11.22 28.46 a 2.08 ab 4.26 10.95 ab
Significance 0.094 0.558 0.097 0.068 0.589 0.004
CV 0.21 0.13 0.20 0.21 0.17 0.24
*= Values followed by different letters in the same treatment and column showed significant differences (DMRT;α
= 0.05). ns= non significantly different (ANOVA; α = 0.05).
Table 5. Classification of resistance criteria of five shallot varieties treated with combination of Bacillus velezensis
B-27 and Bacillus cereus RC76 in sandy field
No. Varieties Disease intensity (%) Resistance criteria
1. Tajuk 6.32 Resistant
2. Bima Brebes 9.43 Resistant
3. Bauji 12.71 Moderately resistant
4. Crok Kuning 11.21 Moderately resistant
5. Manjung 10.95 Moderately resistant
Determination of the resistance category of varieties based on twisted disease intensity in the field at 42 DAP.

velezensis can degrade fungal cell walls by producing indicated the disease progression of twisted disease.
glucanase and peptidoglycan. A study on B. velezensis The AUDPC value was obtained based on the twisted
FKM10 by Wang et al. (2020) showed damage to the disease intensity used to describe the type of plant
cell walls and mycelium of F. verticillioides due to resistance to pathogens quantitatively. Observations
high glucanase production. B. velezensis also forms in the greenhouse showed that the AUDPC of shallots
peptidoglycan which replaces the cell wall, resulting in treated with combination of B. velezensis B-27 and
barriers to fungal nutritional needs and fungal growth. B. cereus RC76 was significantly different from the
Bacillus spp. can also affect the physiological inoculation and non-inoculation treatments (Table 6).
response of plants by inducing plant resistance to disease. The five shallot varieties did not show a significant
Secondary metabolite compounds produced by bacteria difference between varieties in the AUDPC value. The
can recognize and activate the signaling pathways of smallest AUDPC was shown by Bauji with AUDPC of
plant resistance induction. B. cereus RC76 can induce 248.88, which was not significantly different from Tajuk,
jasmonic acid in shallots which act as a signaling Bima Brebes, Crok Kuning and Manjung. The AUDPC of
pathway for plant resistance to twisted disease (Wulan Bauji with inoculation treatment showed that the disease
et al., 2022). B. velezensis produces surfactin, iturin, development in Bauji was slow, and the AUDPC of Bauji
L-hydroanticapsin, Oxydifficidin, and Bacillibactin, after applied with the combination of B. velezensis B-27
which can increase plant growth and induce plant and B. cereus RC76 also showed a lower number than
resistance to pathogens (Ye et al., 2018; Nifakos et al., some other varieties.
2021). Chen et al. (2022) stated that the gene of SIMPK3 The treatment with a combination of B. velezensis
in B. velezensis regulates the accumulation of reactive B-27 and B. cereus RC76 had significant effect in
oxygen species by regulating genes related to pathogen AUDPC or disease progression on five shallot varieties.
pressure and can produce indole acetic acid and surfactin, Tajuk variety treated with a combination of B. velezensis
which prevent and inhibit disease. B-27 and B. cereus RC76 had the lowest AUDPC value,
and the largest was shown by Crok Kuning. The results
Area Under the Disease Progression Curve (AUDPC). indicated that the development of twisted disease on
The AUDPC value from the results of the study Tajuk was the lowest compared to the other four shallot
24 J. Trop. Plant Pests Dis. Vol. 24, No. 1, 2024: 17–27

varieties. This was consistent with the twisted disease AUDPC value. AUDPC describes the progression of
intensity on Tajuk treated with the combination of B. twisted disease based on the disease intensity from 14
velezensis B-27 and B. cereus RC76 in the greenhouse, to 42 DAP. The disease progression in the field showed
which had the smallest number. Based on AUDPC variations among the five shallot varieties. The lowest
overall, Bauji and Tajuk showed the best resistance AUDPC was found at Tajuk variety of 114.92, which was
response among the five shallot varieties against twisted significantly different from cv. Crok Kuning (174.77) and
disease, and there was an increase in resistance response Bauji, which had the highest AUDPC value (Figure 2).
after the treatment with combination of B. velezensis The AUDPC in the Tajuk variety in the field mirrored
B-27 and B. cereus RC76. the results from the greenhouse, where the AUDPC of
The greater the AUDPC value, the lower the Tajuk was the lowest compared to the other varieties.
level of plant resistance to disease. Disease progression, The results indicated that the Tajuk variety had better
as calculated by AUDPC, is frequently used because resistance than Bima Brebes, Bauji, Crok Kuning and
the phenology and growth of the plant host also affect Manjung varieties because it exhibited a slower disease
disease development. The resistance of plant hosts must progression.
be measured by quantitative parameters in the form of A comparison of the AUDPC values of shallot
disease rates when the disease emerges in plants during varieties in the field showed lower results than in the
the growing season, and pathogen infection is not greenhouse because the disease intensity in the field
sustainable but depends on the environment (Jeger & was also lower than in the greenhouse. The slower
Viljanen-Rollinson, 2001). disease development in the field could be attributed to
The results of the field study revealed that the environmental factors that were not conducive to the
varieties treated with the combination of B. velezensis development of pathogens, the number and virulence
B-27 and B. cereus RC76 had a significant effect on the levels of pathogens, and the resistance level of shallot
Table 6. AUDPC of twisted disease on five shallot varieties applied with the combination of Bacillus velezensis
B-27 and Bacillus cereus RC76 in the greenhouse
Varieties
Treatments Average*
Tajuk Bima Brebes Bauji Crok Kuning Manjung
Bacillus spp. + inoculation 217.75 435.54 248.90 451.13 233.38 317.4 b
Inoculation 622.25 497.82 482.18 482.28 575.56 532.02 a
Non-inoculation 0.00 62.25 15.56 15.56 46.69 28.01 c
Average ns
280.00 331.87 248.88 316.32 a 285.21 (-)
The sign (-) indicates that there is no interaction between treatments and varieties. *= Values followed by different
letters in the same treatment and column showed significant differences (DMRT; α= 0.05). ns= non significantly
different (ANOVA; α= 0.05).
200
a a
180 ab
160 ab
140
120 b
100
AUDPC

80
60
40
20
0
Tajuk Bima Brebes Bauji Crok Kuning Manjung
Varieties
Figure 2. Area under the disease progress curve on five shallot varieties applied with a combination of Bacillus
velezensis B-27 and Bacillus cereus RC76 in the field. *= Values followed by different letters in the same
treatment and column showed significant differences (DMRT; α= 0.05).
Pratiwi et al. Response of five shallot varieties applied with Bacillus spp. 25

varieties induced by the combination of B. velezensis ACKNOWLEDGMENTS

B-27 and B. cereus RC76.
AUDPC serves as a standard for classifying The authors extended the gratitude to the
varieties in terms of disease resistance. Jeger & Viljanen- Australian Center for International Agricultural Research
Rollinson (2001) state that AUDPC is the basis for (ACIAR) with project number SLAM/2018/145 which
quantitatively determining the resistance of barley has funded this research.
varieties to leaf rust disease. Various measurements
are involved in the classification of plant resistance, FUNDING
including assessment of the growth phase, calculation
of disease development, and the computation of the area This research is funded and supported by ACIAR-
under the disease progress curve (AUDPC). SLaM/2018/145 (year 2022-2023) project.
Aprilia et al. (2020) analyzed 19 shallot genotypes
with characteristics of resistance to twisted disease based AUTHORS’ CONTRIBUTIONS
on disease incidence, incubation period and disease
infection rate. The dendrogram resulted in three groups of AHP is the first author for the contribution to
resistance categories. Bima Brebes, Bauji, and Manjung performing the research and writing this manuscript. AW
belonged to the susceptible group, which tended to have as the corresponding author considered the experimental
the highest disease incidence and the fastest disease design, as a proofreader and revised this manuscript.
incubation period. Tajuk variety was included in the TJ is providing the bacteria isolates and revised this
moderately resistant group because it showed lower manuscript. AW revised the methods and data analysis.
disease incidence than the susceptible group but higher SS led and supervised the project. The authors provided
than the resistant group and had a slower incubation responses and comments on the research flow, data
period than the susceptible group. analysis, and interpretation as well as the shape of the
Based on the results of field studies, Tajuk and manuscript. All the authors have read and approved the
Bima Brebes varieties showed an increase in resistance final manuscript.
criteria to a resistant category, an increase in the category
level compared to the research by Aprilia et al. (2020). COMPETING INTEREST
The varieties Bauji and Manjung increased to moderately
resistant criteria, while Crok Kuning was not mentioned The authors declared that there is no potential
in the literature and was included in the moderately conflict of interest.
resistant criteria. The differing results of the studies
could be caused by variations in time, treatment, and REFERENCES
environment used for research, resulting in different
levels of resistance shown. Aprilia I, Maharijaya A, & Wiyono, S. 2020. Keragaman
genetik dan ketahanan terhadap penyakit layu
CONCLUSIONS Fusarium (Fusarium oxysporum f.sp cepae)
bawang merah (Allium cepa L. var. aggregatum)
The application of the combination of B. velezensis Indonesia [Genetic diversity and Fusarium
B-27 and B. cereus RC76 had a significant effect on the wilt disease resistance (Fusarium oxysporum
incubation period, incidence, and intensity of twisted f.sp. cepae) of Indonesian shallots (Allium
disease in five shallot varieties. The combined treatment cepa L. var aggregatum). Jurnal Hortikultura
of B. velezensis B-27 and B. cereus RC76 reduced the Indonesia. 11(1): 32–40. https://doi.org/10.29244/
incidence of twisted disease by up to 70% and the jhi.11.1.32-40
disease intensity by up to 87%. The five shallot varieties Cahyaningrum H, Suryanti, & Widiastuti A. 2020.
showed different and increased resistance responses to Response and resistance mechanism of shallot var.
twisted disease, with Tajuk and Bima Brebes classified as Topo, a North Molluca’s local variety against basal
resistant, while Bauji, Crok Kuning, and Manjung were rot disease. Proceedings of the 5th International
classified as moderately resistant. Overall, Tajuk variety Conference on Food, Agriculture and Natural
treated with the combination of B. velezensis B-27 and Resources (FANRes 2019). pp. 71–75. https://doi.
B. cereus RC76 showed the best results in suppressing org/10.2991/aer.k.200325.015
twisted disease.
Chen Q, Qiu Y, Yuan Y, Wang K, & Wang H. 2022.
26 J. Trop. Plant Pests Dis. Vol. 24, No. 1, 2024: 17–27

Biocontrol activity and action mechanism of Le D, Audenaert K, & Haesaert G. 2021. Fusarium basal
Bacillus velezensis strain SDTB038 against rot: profile of an increasingly important disease
Fusarium crown and root rot of tomato. Front. in Allium spp. Trop. Plant Pathol. 46: 241–253.
Microbiol. 13: 994716. https://doi.org/10.3389/ https://doi.org/10.1007/s40858-021-00421-9
fmicb.2022.994716
Lestiyani A, Wibowo A, & Subandiyah S. 2021.
Djaenuddin N, Muis A, & Nonci N. 2018. Screenhouse Pathogenicity and detection of phytohormone
test of eight biopesticide formulation Bacillus (gibberellic acid and indole acetic acid) produced
subtilis against downy mildew, Peronosclerospora by Fusarium spp. that causes twisted disease in
philipinensis, on corn plant. J. Trop. Plant Pests shallot. Jurnal ProteksiTanaman. 5(1): 24–33.
Dis. 18(1): 51–56. https://doi.org/10.23960/j. https://doi.org/10.25077/jpt.5.1.24-33.2021
hptt.11851-56
Maharijaya A, Harti H, Nuryana FI, Rosyidin C, Suryo,
Emeliawati, Salamiah, & Fitriyanti D. 2022. Pengendalian Helmi, Sulistyono A, & Akat. 2016. Description
penyakit moler (Fusarium oxysporum) pada of Tajuk Shallot Variety. Agriculture Department
bawang merah dengan serbuk kulit jengkol of Nganjuk District. https://hortikultura.pertanian.
(Pithecellobium jiringa) di lahan gambut [Control go.id/. Accessed 20 February 2023.
of twisted disease (Fusarium oxysporum) on
Nifakos K, Tsalgatidou PC, Thomloudi EE, Skagia A,
shallots with jengkol bark powder (Pithecellobium
Kotopoulis D, Baira E, Delis C, Papadimitriou K,
jiringa) in peatlands]. Proteksi Tanaman Tropika.
Markellou E, Venieraki A, & Katinakis P. 2021.
5(2): 499–505. https://doi.org/10.20527/jptt.
Genomic analysis and secondary metabolites
v5i2.1255
production of the endophytic Bacillus velezensis
Hadiwiyono, Sari K, & Poromarto SH. 2020. Yields losses Bvel1: A biocontrol agent against Botrytis cinerea
caused by basal plate rot (Fusarium oxysporum causing bunch rot in post-harvest table grapes.
f.sp. cepae) in some shallot varieties. Caraka Plants. 10(8): 1716. https://doi.org/10.3390/
Tani: Journal of Sustainable Agriculture. 35(2): plants10081716
250–257. https://doi.org/10.20961/carakatani.
Ntushelo K, Ledwaba LK, Rauwane ME, Adebo OA, &
v35i2.26916
Njobeh PB. 2019. The mode of action of Bacillus
Herlina L, Istiaji B, & Wiyono S. 2021. The causal species against Fusarium graminearum, tools for
agent of Fusarium disease infested shallots investigation, and future prospects. Toxins. 11(10):
in Java Islands of Indonesia. E3S Web of 606. https://doi.org/10.3390/toxins11100606
Conferences. 232: 03003. https://doi.org/10.1051/
Prakoso EB, Wiyatingsih S, & Nirwanto H. 2016.
e3sconf/202123203003
Uji ketahanan beberapa kultivar bawang merah
Ilmiah HH, Sulistyaningsih E, & Joko T. 2021. Fruit (Allium ascalonicum) terhadap infeksi penyakit
morphology, antioxidant activity, total phenolic moler (Fusarium oxysporum f.sp. cepae)
and flavonoid contents of Salacca zalacca [Endurance test on different cultivars shallots
(Gaertner) Voss by applications of goat manures (Allium ascalonicum) against infectious diseases
and Bacillus velezensis B-27. Caraka Tani: moler (Fusarium oxysporum f.sp. cepae)].
Journal of Sustainable Agriculture. 36(2): Plumula. 5(1): 10–20.
270–282. https://doi.org/10.20961/carakatani.
Pusat Data dan Sistem Informasi Pertanian (Pusdatin).
v36i2.43798
2013. Workshop Hasil Pengembangan Metode
Jeger MJ & Viljanen-Rollinson SLH. 2001. The use Konversi Bawang Merah [Workshop of
of the area under the disease-progress curve Convertion Method Development Result of
(AUDPC) to assess quantitative disease resistance Shallot]. Kementrian Pertanian. Jakarta.
in crop cultivars. Theor. Appl. Genet. 102(1):
Rabbee MF, Ali MDS, Choi J, Hwang BS, Jeong SC, &
32–40. https://doi.org/10.1007/s001220051615
Baek KH. 2019. Bacillus velezensis: A valuable
Khan N, Maymon M, & Hirsch AM. 2017. Combating member of bioactive molecules within plant
Fusarium infection using Bacillus-based microbiomes. Molecules. 24(6): 1046. https://doi.
antimicrobials. Microorganisms. 5(4): 75. https:// org/10.3390/molecules24061046
doi.org/10.3390/microorganisms5040075
Rahma AA, Suryanti, Somowiyarjo S, & Joko T. 2020.
Pratiwi et al. Response of five shallot varieties applied with Bacillus spp. 27

Induced disease resistance and promotion of four shallot (Allium cepa L. Aggregatum Group)
shallot growth by Bacillus velezensis B-27. cultivars to moler disease (Fusarium spp.) after
Pak. J. Biol. Sci. 23(9): 1113–1121. https://doi. bulb treatment. Acta Hortic. 1143: 69–76. https://
org/10.3923/pjbs.2020.1113.1121 doi.org/10.17660/ActaHortic.2016.1143.11
Ramírez V, Martínez J, Bustillos-Cristales MDR, Wijoyo RB, Sulistyaningsih E, & Wibowo A. 2020.
Catañeda-Antonio D, Munive JA, & Baez A. Growth, yield and resistance responses of three
2022. Bacillus cereus MH778713 elicits tomato cultivars on true seed shallots to twisted disease
plant protection against Fusarium oxysporum. J. with salicylic acid application. Caraka Tani:
Appl. Microbiol. 132(1): 470–482. https://doi. Journal of Sustainable Agriculture. 35(1): 1–11.
org/10.1111/jam.15179 https://doi.org/10.20961/carakatani.v35i1.30174
Rohma M & Wahyuni WS. 2022. Pengendalian penyakit Wiyatiningsih S. 2021. A study on twisting disease
layu Fusarium oxysporum f.sp cepae pada epidemic on shallot. AcademiaLetters. 1516.
tanaman bawang merah dengan air rebusan serai https://doi.org/10.20935/al1516
dapur (Cymbopogon citratus) [Control of wilt
Wiyatiningsih S, Wibowo A, & Triwahyu E. 2009.
disease (Fusarium oxysporum f.sp. cepae) on
Tanggapan tujuh kultivar bawang merah terhadap
shallots with lemongrass (Cymbopogon citratus)
infeksi Fusarium oxysporum f. sp. cepae penyebab
boiled water. Berkala Ilmiah Pertanian. 5(2):
penyakit moler [Responses of seven shallot
65–69. https://doi.org/10.19184/bip.v5i2.28856
cultivars to infection of Fusarium oxysporum f.sp.
Simko I & Piepho HP. 2012. The area under the disease cepae cause of moler disease. Jurnal Pertanian
progress stairs: Calculation, advantage, and MAPETA. 12(1): 7–13.
application. Phytopathology. 102(4): 348-451.
Wulan EIR, Wibowo A, Joko T, & Widiastuti A. 2022.
https://doi.org/10.1094/PHYTO-07-11-0216
Induced resistance mechanism of twisted disease
Sudana M & Lotrini M. 2005. Pengendalian terpadu suppression of shallot by Bacillus spp. Jurnal
penyakit layu (Ralstonia solanacearum Smith) Perlindungan Tanaman Indonesia. 26(1): 40–50.
dan nematoda puru akar (Meloidogyne spp.) pada https://doi.org/10.22146/jpti.73198
tanaman jahe gajah. J Trop Plant Pests Dis. 5(2):
Ye M, Tang X, Yang R, Zhang H, Li F, Tao F, Li F, &
97–103. https://doi.org/10.23960/j.hptt.2597-103
Wang Z. 2018. Characteristics and application of
Wang C, Zhao D, Qi G, Mao Z, Hu X, Du B, Liu K, a novel species of Bacillus: Bacillus velezensis.
& Ding Y. 2020. Effects of Bacillus velezensis ACS Chem. Biol. 13(3): 500–505. https://doi.
FKM10 for promoting the growth of Malus org/10.1021/acschembio.7b00874
hupehensis Rehd. and inhibiting Fusarium
Zhou H, Ren ZH, Zu X, Yu XY, Zhu HJ, Li XJ, Zhong J, &
verticillioides. Front. Microbiol. 10: 2889. https://
Liu EM. 2021. Efficacy of plant growth-promoting
doi.org/10.3389/fmicb.2019.02889
bacteria Bacillus cereus YN917 for biocontrol of
Wibowo A, Kaeni E, Toekidjo T, Subandiyah S, rice blast. Front. Microbiol. 12: 684888. https://
Sulistyaningsih E, & Harper S. 2016. Responses of doi.org/10.3389/fmicb.2021.684888