The use of biological agents in controlling diseases of shallots for tss production

Stemphylium disease caused by Stemphylium vesicarium is an important disease that becomes an obstacle in TSS production. The disease attacks can lead to seed harvest failure. In the field. Stemphylium disease is associated with purple spot disease caused by the fungus Alternaria porri. The purpose of this study was to obtain biological agents that were effective in controlling S vesicarium and A porri on TSS production. The tests were carried out in-vitro in the laboratory and in planta in the field under a plastic shade. The experimental design used a randomized block design with 11 treatments (Bacillus sp.1. Bacillus sp. 3., Pseudomonas florescens (Pf)., Trichoderma sp.2., Trichoderma sp.3., Bacillus sp.1 + Trichoderma sp.2 + Pf., Bacillus sp.3 + Trichoderma sp.2 + Pf., Bacillus sp.1 + Trichoderma sp.3 + Pf., Bacillus sp.3 + Trichoderma sp.3 + Pf, and Control). Each treatment was repeated 3 times. The results showed that the biological agents Trichoderma sp., Bacillus sp., and Pseudomonas fluorescent as antagonist agents have varying inhibitory abilities against pathogens Stemphylium vesicarium and Alternaria porri. Inhibition by Bacillus sp. against S. vesicarium by 60.51-63.75% and by Trichoderma sp. by 25.78-31.47%. Meanwhile for A. porri. both bacterial and fungal antagonist agents had an inhibition between 45.09-79.15%. Antagonist agents Trichoderma sp.3 alone and the combination of Bacillus sp.1 + Trichoderma sp.3 + Pseudomonas fluorescens can increase the number of leaves and the number of seedlings of shallots. Antagonist agent Trichoderma sp.3 alone and in combination between Bacillus sp.3 + Trichoderma sp.3 + P. fluorescens can reduce the attacks intensity of S. vesicarium and A. porri diseases.


Introduction
Shallot (Allium ascalonicum L.) is a vegetable commodity that is quite important and popular in Indonesia, has high economic value, has functions as a flavor enhancer, and can be used as an ingredient in traditional medicine.The prospect of developing shallots is promising, which is indicated by the increasing consumption of shallots in line with the increase in population.The average increase in per capita shallot consumption per year from 2005 to 2019 fluctuated with an increasing trend of reaching 2.67% [1].In addition, this commodity is one of the seven strategic agricultural commodities, because it affects farmers' lives, the macro economy, and the inflation rate.At the Cabinet meeting for the Food 1230 (2023) 012098 IOP Publishing doi:10.1088/1755-1315/1230/1/012098 2 Sector in Bukittinggi on 29 October 2013, the government determined shallots as a strategic food commodity.
Shallot farming is a business that has a high risk.There are a few challenges and obstacles encountered in cultivation, such as pest attacks that can derail harvests.Pest attacks that appear throughout the year on shallot plantations will reduce productivity.Pest attacks will increase during the dry season while disease attacks generally increase during the rainy season.
Nationally, the needs of the population who consume shallots are around 80% or 816,960 tons, and other needs are 122,544 tons so that the total need for shallots in 1 year is around 939,504 tons.So that the average need every month is 78,292 tons.This need has been able to be met with the productivity produced by farmers, namely the national average of 10 t/ha [2] The per capita need is 4.56 kg per year or 0.38 kg/month, but ahead of religious holidays it will increase by 10-20% [3].
The problem faced in increasing productivity is the limited availability of seeds each planting season arrives.The seeds used generally come from tuber seeds that have been stored for 2-3 months, so they require costs for maintenance while in storage.As a substitute for using seeds from tubers, shallot seeds from botanical seeds (true seed shallot/TSS) have been produced.Indonesian Vegetable Research Institute (IVEGRI) has provided the technology for TSS seed production but there are still several obstacles, including pest attacks and diseases.Yield loss in shallot plants can be caused by infection with several diseases during the growing period of the crop [4].Stemphylium leaf blight (SLB) disease is a serious disease that attacks leaves, petioles, and flower stalks which will produce seeds [5,6].Stemphylium disease associated with Alternaria porri is a major disease that can attack flower stalks resulting in broken and rotted flower stalks so that the flowers do not produce capsules.Stemphylium leaf blight is considered less important in plant diseases that attack shallot leaves, but if the attack intensity is high it can affect production [7].
Control of Stemphylium disease has been widely carried out.including using technical and chemical cultures [8,9].Biological control using microorganisms has been proven successful in controlling various plant diseases in many countries [10][11][12].The plant resistance induction method is a method that has the potential to reduce the severity of a plant pathogen disease and can increase the level of resistance to subsequent attacks [13,14].The inducer can be useful beneficial microorganisms or certain chemicals such as salicylic acid and asibenzolar-s-methyl-mankozeb which induce systemic resistance (SAR) in plants [15].
In Indonesia, there has not been much research on controlling Stemphylium disease in shallot, especially on TSS production.IVEGRI has a collection of biological agents that have not been used to control Stemphylium disease in shallots in producing TSS.This study aimed to obtain biological agents that effectively control the S. vesicarium and A. porri fungi on TSS production.

Materials and methods
The research was carried out from February to December 2020 at the Laboratory and Margahayu Experimental Garden.Indonesian Vegetable Research Institute in Lembang at an altitude of 1250 m asl.The material used in this study was Stemphylium sp. and Alternaria porri, Trichoderma sp.

In planta testing.
The shallots used were the Trisula variety which had become seeds, stored for 2 months, and had been vernalized for one month at 10°C in cool storage.Seeds were planted in polybags measuring 40 x 35 cm with a planting medium mixed with chicken manure, soil, and husk charcoal (1:1:1).The TSS production method [16] which is outlined in the TSS Production Manual.The treatment is the use of biological agents Trichoderma sp., Bacillus sp., and Pseudomonas fluorescent as antagonist biological agents, either alone or in the combination of the three.As for the treatment is:

Pseudomonas florescens -
The treatment applied to medium is by sprinkling 30 ml of solution taken from inoculum with 10 ml/L concentration.The experimental design used was a randomized block design with ten treatments and three replications.Each treatment consisted of 5 polybags and 3 shallot seed bulbs which were planted in each polybag.

Observation parameters.
In the in vitro test, the first observation was made one day after inoculation (DAI), while follow-up observations were made every day until 7 DAI.For testing biological agents, observations were made by measuring the radius of the pathogen colony.The results of inhibition measurements are calculated using formula [17] as follows: P = Percentage of inhibition R1 = Radius of the pathogen away from the antagonist R2 = Radius of the pathogen toward the antagonist In planta testing was carried out on attack symptoms that appeared on leaves and flower stalks.Observation of symptoms was carried out using the following scoring system: 0 = no attack, 1 = 10% of affected leaves, 2 = 20-30% of affected leaves, 3 = 40-50% of affected leaves, 4 = 60-70% of affected leaves, 5 = >70% [18].The degree of damage to leaves is determined by the formula: Observations were made from two weeks after planting, with an interval of one week.Plant sampling was carried out with a total sample of 10 plants.In addition, it is also observed for plant growth (leaf length, the number of leaves, and the number of tillers).
2.1.4.Data analysis.The quantitative data obtained were analyzed using variance, if significantly different they were further tested by Duncan DMRT's multiple range test at 5% significance level using SAS version 9.1.3(SAS Institute.1990).

Antagonist test of biological agents against pathogenic fungi S. vesicarium and A. porri in vitro.
To determine the percentage of inhibition of antagonistic agents against S. vesicarium and A. porri pathogens before being applied to shallot plants, an in vitro test was carried out in the laboratory.Based on in vitro tests carried out on petri dishes, it was found that all the antagonist agents tested could inhibit pathogen isolates with various inhibition percentages.The percentage of inhibition of antagonistic agents against S. vesicarium disease is shown in Table 2 and the results of dual culture on petri dishes (Figure 1).In the first observation after the double test, it appears that the bacterial antagonist agent Bacillus sp.1 produced the highest percentage of inhibition reaching 50%, followed by Bacillus sp.3 (35.44%) and Bacillus sp.2.(24.69%), this inhibition was higher than the Trichoderma antagonists from the three species, and statistically significantly different between the antagonists from bacteria and the antagonists from fungi.This condition continued until the seventh day of observation.with high inhibition produced antagonists of bacterial origin.While the double test of antagonistic agents with A. porri obtained different results with Stemphylium, on the first day of observation the percentage of Trichoderma inhibition of the three species ranged from 26.23-34.09%higher than the antagonist agent Bacillus of the three species (Table 3) and Figure 2.   of this multiple tests, it can be discussed as a whole that the growth of each pathogenic isolate (Stemphylium and Alternaria) was affected by the growth of antagonist agents with varying inhibition, which was shown by the growth rate of pathogenic fungi with different growth characteristics.Likewise with the antagonist agents used, such as Bacillus sp. which has a mechanism that secretes antibiotic chemical compounds and bacteriolytic enzymes [19].According [20] , the antagonist agent B. subtilis produces several antibiotic compounds such as bacitracin, basilin, bacillamycin, diphydin, oxidifisidin, lecithinase, and subtilisin.Besides that, it can also produce hydrolytic enzymes such as glucanase or protease and lipopeptide antibiotics surfactin, Fengycin, and iturin [21].Meanwhile.according [22], the mechanism of Trichoderma is mycoparasitism, antibiotics, competition, releasing enzymes, and inducing resistance.Trichoderma harzianum produces secondary metabolites in the form of lytic enzymes such as chitinase, glucanase, and protease, which degrade the cell wall of the fungus.The hyphae of T. harzianum will entwine the hyphae of the pathogenic fungus so that the hyphae of the pathogenic fungus will undergo vacuolation, lysis, and finally disintegrate.After that.T. harzianum penetrated and used the hyphal contents of pathogenic fungi as a food source [23,24].

In planta testing
3.2.1.Plant growth.Observation of plant growth was carried out twice, on 21 days after planting (DAP) and 35 DAP, with the parameters of leaf length, the number of leaves, and the number of tillers.The parameter observations were carried out to determine the effect of giving antagonist agents as a growth promoter after application.The average yield of shallot growth during the application of antagonist agents is shown in Figures 3a, 3b and 3c.Based on statistical tests on the three parameters observed after the application of antagonist agents there was no significant difference, but if seen from the numerical average using the treatment biological agents showed a higher number of leaves and tillers than the control, except that the leaf length was relatively evenly distributed, with an average of 34-37 cm at 21 DAP and 47-50 cm at 35 DAP.The average number of leaves at 21 DAP ranged from 32-37 leaves and only 27 leaves in the control.At the age of 35 DAP ranged from 43-53 leaves and the control 36 leaves.As for the number of tillers at the age of 21 DAP ranged from 6-8 and the control only 5 tillers.At the age of 35 DAP the number of tillers in the antagonistic agent treatment ranged from 7-10, but only 6 in the control.Regarding the ability of microorganisms to stimulate growth according to research by [25] that wheat plants that were applied antagonistic agents (Aspergillus niger and T. hamatum) had a significant effect on growth and yield in all treatments compared to controls grown in two growing seasons.

Disease attack intensity.
The first observation of disease symptoms was carried out after two applications of the antagonist agent, when the shallot plants were 21 DAP.Symptoms of the disease that appear are A. porii.S. vesicarium, and C. gloeosporioides.The average attack intensity of A. porii ranged from 3.11-10.66%(Table 4).The attack intensity observed in the plant samples increased with the age of the plants.but statistically.there was no significant difference between these treatments.The attack symptoms observed up to 56 DAP were still on the leaves with an average attack intensity of 21.67-29.78%.The lowest attack intensity was found in the combination treatment of Bacillus sp.3 + Trichoderma sp.3 + Pf (21.67%) and the highest attack intensity was in the control (29.78%).
The symptoms caused by the fungus S. vesicarium have relatively the same attack intensity as those of A. porii.The symptoms of S. vesicarium and A. porii are often found to be associated with one symptom [26].In this study, the symptoms of S. vesicarium were only found in shallot plants aged 28 DAP (Table 5) with relatively lower attack symptoms than A. porii.At the age of 28 DAP, the average attack intensity ranged from 0.89-4.00%.However, after the shallot plant was 56 DAP the intensity of the attack symptoms increased, with an average low attack symptom resulting in the treatment applied showed the lowest attacks of A. porii and S. vesicarium.This is consistent with research [27] that the application of B. subtilis alone or in combination with T. harzianum T5 can suppress Fusarium oxysporum with disease incidence 4.66% and disease severity 1.09%.In addition.Trichoderma can release elicitors that can induce plants to release various types of signals such as salicylic acid, and jasmonic acid which can trigger the expression of defense proteins [28].Trichoderma can also suppress Fusarium wilt disease [29].In addition to these two diseases, other symptoms of the disease that appear are anthracnose (C.gloeosporioides).In the first observation of plant age, 21 DAP symptoms of attack intensity ranged from 1.33 to 6.67% (Table 6).Observations at the age of 28 DAP and 35 DAP of attack symptoms increased.but at the age of 42 DAP, the symptoms of the attack began to decrease in almost all treatments except for the Bacillus sp.3 treatment.From the results of these observations, it seems that the treatment applied to Pseudomonas fluorescence showed the lowest intensity (9%), while other treatments ranged from 10.33-18.33% at plant age 56 DAP.The effect of biological agents on the number of flowers, umbel weight, and the number of capsules per umbel can be seen in table 7. Treatment of Trichoderma sp.isolates.3 produced the highest number of capsules per umbel, while the number of flowers and umbel weight were almost evenly distributed in each treatment.The capsule is a fruit term for the genus Allium, the average capsule is 52.4-66.6 [30] and 25 (without BAP and Boron) and 43 (with BAP and Boron) [31].In this activity, the number of capsules was higher (85.11-119.22), but the high number of capsules, in this case.was still mixed between empty and filled ones.
severity n = The number of plants observed for each attack category v = Scale value of each attack category N = Number of plants observed V = Scale value of the highest attack category 1230 (2023) 012098 IOP Publishing doi:10.1088/1755-1315/1230/1/0120984

Figure 3 .
Figure 3. Development of leaf length (a), number of leaves (b) and number of tillers (c) on shallot.1.

by
Trichoderma sp.3 (13.78%) and Bacillus sp.3 + Trichoderma sp.3 + Pf (15.11%).Treatment of antagonistic agents alone or in combination can suppress disease attacks, as in this study the single treatment of Trichoderma sp.3 and the combination treatment of Bacillus sp.3 + Trichoderma sp.3 + Pf

1 .
Trichoderma sp.3 alone and in combination between Bacillus sp.1 + Trichoderma sp3 + Pseudomonas fluorescens can increase the number of leaves and the number of shallot tillers.2. Trichoderma sp.3 alone and in combination between Bacillus sp3 +Trichoderma sp3+P.fluorescens can reduce the intensity of S. vesicarium and A. porri disease attacks.

Table 4 .
The effect of several biological agents alone and in combination on the intensity of Alternaria porii attacks.DAP = days after planting, ** The numbers in the columns followed by the same letters are not significantly different according to Duncan Multiple with α = 5%

Table 5 .
The effect of several biological agents alone and in combination on the intensity of S. vesicarium attacks.

Table 6 .
The effect of several biological agents alone and in combination on the intensity of C. gloeosporioides attacks.DAP = days after planting, ** The numbers in the columns followed by the same letters are not significantly different according to Duncan Multiple with α = 5%

Table 7 .
Effect of biological agents on the number of flowers.umbelweight.and number of capsules per umbel.) The numbers in the columns followed by the same letters are not significantly different according to Duncan Multiple with α = 5% *