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TOMATO (LYCOPERSICUM ESCULENTUM MILL.) RESILIENCE ENHANCEMENT WITH INDIGENOUS ENDOPHYTIC BACTERIA AGAINST BEMISIA TABACI (HEMIPTERA: ALEYRODIDAE).

Byline: H. Hamid, Y. Yanti, F. R. Joni and Nurbailis

KeyWords: Bemisia tabaci, development stage, endophytic bacteria, resistance, tomato.

INTRODUCTION

B. tabaci is an important pest on tomato plants. B. tabaci's attack can cause yield loss to range from 20-100% (Setiawati et al. 2007). B. tabaci causes damage to plants in two ways. Directly effect as a result of their eating activities, that is the closure of the stomata by the honeydew released by the nymph, formation of chlorotic spots on the leaves as a result of damage to part of the tissue due to stethicle puncture, and falling leaves and can inhibit growth plants (DeBarro 1995). A secondary effect, because they act as essential vectors of virus diseases. There are 100 types of viruses that can be transmitted by B. tabaci (Byrne and Bellows 1990). Control measures taken to overcome B. tabaci's attacks include mechanical control, technical culture, planting resistant varieties, and spraying insecticides. The farmers are more likely to use synthetic pesticides because they are more practical and easy to apply. Improper use of insecticides can have adverse effects on the environment.

The negative effects caused by pesticides include water, soil, and air pollution, the emergence of resistant pest species, the emergence of new pest species or the explosion of secondary pests, resurgence, damaging the balance of ecosystems, and the impact on public health (Adriyani, 2006). To avoid the harmful effects of insecticides, environmentally friendly controls can be used, namely by using biocontrol biological agents, one of which is Plant Growth Promoting Rhizobacteria (PGPR). The presence of bacteria acting as PGPR in plants can be grouped according to their place of colonization, namely rhizosphere, rhizoplane, and endophytes (Soesanto, 2008). Endophytic bacteria are microorganisms that live and colonize host tissues without causing adverse effects. Endophytic bacteria have been used in controlling plant pathogens (Marwan et al. 2011; Munif et al. 2015).

Endophytic bacteria are also able to induce plant resistance to suppress the development and cause the death of plant pests (Rajendran et al. 2011; Pineda et al. 2012; Praca 2012; Munif et al. 2015; Utami 2018). The results of the study by Yanti et al. (2017) obtained eight isolates of indigenous endophytic bacteria from tomato plants which were able to suppress the development of Fusarium oxysforum and Ralstonia syzyggii in planta. These isolates need to be tested in increasing the resistance of tomato plants to B. tabaci attacks. The purpose of this study was to obtain the best endophytic bacteria isolates in improving the resilience of tomato plants to B. tabaci

MATERIALS AND METHODS

Study area: The study was conducted at the Microbiology and Greenhouse Laboratory, Faculty of Agriculture, Universitas Andalas, West Sumatera from January to July 2019.

Methodology: The research was the experimental method used a completely randomized design (CRD) with10 treatments and three replications. The treatment consisted of 8 isolates of IEB (see Table 1), positive controls (Control) (without using insecticides), and negative controls (Control N) (using insecticides). For negative control, insecticides were applied by hand sprayer from 1 WAP (Week After Planting) with intervals were one week. The parameters observed were the number of individuals who survived per day and the length of days needed to develop per stage.

Procedures

Rejuvenation and multiplication of endophytic bacterial isolates: The isolates of indigenous endophytic bacteria were obtained from Yanti's collection (2017). IEB isolates were rejuvenated using one bacterial ose transferred to the NA medium in a Petri dish with a scratch method and incubated at room temperature for 2 x 24 hours. The multiplication of endophytic bacteria was conducted in 2 stages, namely: (1) pre-culture, one indigenous endophytic bacteria colonies from pure culture were transferred into 10 ml NB medium in a culture bottle and incubated on a rotary shaker at 150 rpm for 24 hours at room temperature. (2) Main culture, 1 ml of the suspension from preculture was transferred to 25 ml of sterile coconut water in a bottle of culture and incubated in the same manner for 3 x 24 hours (Habazar et al. 2007).

The suspension of endophytic bacteria from the main culture was determined by population density based on a comparison with a scale 8 McFarland solution (BaCl 0.8 g + H_2 [SO 44 1% 9.2 g) (bacterial population density estimated at 108cell/ml) (Klement et al. 1990)

Inoculation of Indigenous Endophytic Bacteria (IEB) isolates: The inoculation of endophytic bacteria isolates was conducted for two times, namely in tomato seeds and seedling.

Introduction of B. tabaci on tomato plants: B. tabaci was founded from tomato and chili plants around the Padang. B. tabaci which have been obtained was rearing in a wooden box measuring 60 x 75 x 100 cm3 and covered with gauze. B. tabaci was maintained in tomato plants until imago was formed. Imago formed was then used in endurance testing. Each experimental treatment has inserted a pair of imago B. tabaci on plants aged 1 WAP which had been given a plastic-coated cage on each side with the top coated with gauze.

Data analysis: The parameters observed were the number of individuals who survived per day and the length of days needed to develop per stage. Data were analyzed by analysis of variance with 5% level, if there was a difference, it was followed by a Least Significance Different Test (LSD) at the level of 5%.

Table 1. Name and origin of selected indigenous endophytic bacterial (IEB) isolates.

Isolates###Source of isolate

code

EPL 1.1.3###Padang Lua, Agam, West Sumatera

EPL 1.1.4###Padang Lua, Agam, West Sumatera

EI.AB 2.1###Aia Batumbuak, Solok, West Sumatera

EI.AB 1.2###Aia Batumbuak, Solok, West Sumatera

SNE 2.2###Sungai Nanam, Solok, West Sumatera

TLE 1.1###Taluak, Agam, West Sumatera

TLE 2.3###Taluak, Agam, West Sumatera

EKL 3.3###Koto Laweh, Solok, West Sumatera

Table 2. The average number of B. tabaci individuals per stage in several isolates of indigenous endophytic bacteria on tomato plants.

Treatments###Development Stadia (individual)

###Egg###Nymph###Nymph###Nymph###Pupae###Imago

###instar 1###instar 2###instar 3

EI.AB 1.2###7,667###abcd###6,333###abcd###4,000###ab###1,667###b###0,667###de###0,333###cd

EI.AB 2.1###7,000###bcd###4,333###cd###0,667###c###0,000###c###0,000###e###0,000###d

EPL 1.1.3###6,667###cd###5,667###bcd###3,333###bc###2,333###b###2,667###abcd###2,333###abc

EPL 1.1.4###5,667###D###3,667###d###3,333###bc###2,667###b###2,000###bcde###2,000###bc

KLE 3.3###5,667###D###4,667###bcd###3,667###ab###1,333###bc###67###de###0,333###cd

Control N###10,333###abc###9,333###abc###4,000###ab###3,000###ab###1,667###cde###0,667###cd

SNE 2.2###6,667###cd###6,333###abcd###3,667###ab###1,333###bc###1,000###de###0,333###cd

TLE 1.1###12,667###ab###10,000###ab###9,000###a###6,667###a###4,667###abc###4,333###ab

TLE 2.3###13,333###A###13,667###a###10,667###a###8,000###a###6,333###a###4,333###b

Control###13,000###A###12,000###a###9,333###a###7,333###a###5,667###ab###5,000###a

Table 3. The average number of days needed by B. tabaci to develop on several isolates of indigenous endophytic bacteria to tomato plants.

Treatments###Development Stadia (day)

###Egg###Nymph instar 1###Nymph instar 2###Nymph instar 3###Pupae

EI.AB 1.2###7,3+-0,58###3,3+-0,58###2,6+-0,58###2,3+-0,58###1,6+-2,89

EI.AB2.1###7,6+- 0,58###1,3+-2,31###1+-1,73###0,6+- 1,1531###1,3 +-2,31

EPL 1.1.3###7+-1,00###2,6+-2,31###2+-1,73###1,3+-1,15###3+-2,65

EPL 1.1.4###7,3+-1,15###2+-1,73###1,6+-1,53###1,3+-1,15###3+-2,65

KLE 3.3###7,3+-0,58###3+-0###3+-0###1,3+-1,15###3+-2,65

Control N###7+-1###3,3+-0,58###2,6+-0,58###2,6+-0,58###4,3+-0,58

SNE 2.2###7+-1###3,6+-0,58###2,6+-0,58###2,6+-0,58###5+-0

TLE 1.1###7,3+-0,58###3,3+-0,58###3+-0###2,3+-0,58###4+-0

TLE 2.3###7+-1###4+-0###2,6+-0,58###3+-0###4,6+-0,58

Control###7,3+-0,58###3,6+-0,58###2,6+-0,58###2+-0###4,6+-0,58

Table 4. The survivorship of B. tabaci which was introduced by several indigenous endophytic bacterial isolates and controls.

Treatments###Development Stadia (proportion)

###Egg###Nymph instar 1###Nymph instar 2###Nymph instar 3###Pupae###Imago

EI.AB.1.2###1###0,818###0,519###0,221###0,09###0,039

EI.AB.2.1###1###0,614###0,100###0,000###0,000###0,000

EPL 1.1.3###1###0,850###0,493###0,403###0,34###0,299

EPL 1.1.4###1###0,649###0,579###0,474###0,35###0,351

KLE 3.3###1###0,824###0,632###0,228###0,12###0,053

Control N###1###0,902###0,388###0,291###0,17###0,068

SNE 2.2###1###0,954###0,561###0,197###0,15###0,045

TLE 1.1###1###0,793###0,714###0,532###0,37###0,341

TLE 2.3###1###0,809###0,764###0,571###0,45###0,307

Control###1###0,923###0,715###0,562###0,44###0,385

RESULTS AND DISCUSSION

Result: Based on experiments conducted with a pair of B.tabaci placed on tomato plants that have been introduced with eight isolates of indigenous endophytic bacteria (IEB) showed a significantly different effect on the number of individuals produced with controls without treatment and control sprayed with insecticides. The number of B. tabaci individuals on tomato plants introduced by IEB can be seen in Table 2. The number of eggs placed by B. tabaci on plants introduced by IEB isolates was significantly different than those of controls. Plants that were introduced SNE 2.2 isolates and controls were the plants with the highest number of eggs, which averaged 13-grain crops, while the plants that were introduced were EPL 1.1.4 and KLE 3.3 isolates with the lowest quantity of eggs, which averaged five eggs. In addition to influencing the number of individuals, IEB isolates also affect the amount of B. tabaci individuals who can develop into an imago.

Plants introduced by IEB isolates showed a markedly different development compared to controls. Introduction of EI.AB.2.1 Isolates, KLE 3.3, SNE 2.2, EI.AB 1.2 and control with insecticides shows the development of B. tabaci least developed, whereas control plants show the most developed B. tabaci. EI.AB 2.1 isolate is the best isolate in suppressing the development of B. tabaci. The length of the day required by B. tabaci to develop every stage is not significantly different. The plants that were introduced were EI.AB 2.1 isolates lacking the production of the eggs to become the imago for the longest, namely 23 days, while the treatment without treatment, control with insecticides and EPL 1.1.3 needed the fastest time, 19 days. The time required by B. tabaci to develop each stage (eggs, nymph instar 1, nymph instar 2, nymph Instar 3, pupae and imago) in plants introduced by endophytic bacteria indigenous and controls can be seen in Table 3

The proportion of B. tabaci individuals who survived (Lx) in each stage of development can be seen in Table 4. Based on the calculation of Lx, the proportion of B. tabaci individuals in the nymph 1 phase which showed the highest value was the treatment of SNE 2.2 isolates with Lx 0.954 and the lowest is EI.AB 2.1 with Lx 0.614. In the 2nd instar nymph stage, the proportion of individuals who survived the highest was obtained in the treatment of TLE 2.2 with Lx 0.764, and the lowest was treatment with isolates EI.AB 2.1 with Lx 0.100. In the instar three nymph stadia, the proportion of individuals survived the highest was obtained in the control treatment with a value of Lx 0.562 and the lowest Lx value was found in the isolate EI.AB.2.1 with a value of Lx 0,000.

The highest Lx value of pupae development was observed in TLE 2.2 treatment with a value of 0.450 and the lowest treatment with isolates EI.AB 2.1 with an amount of 0,000. At the imago stage, the highest Lx value was found in control with Lx value of 0.385, and the lowest was in the treatment with isolates EI.AB 2.1 with a value of Lx 0,000. From all stages of development, based on the value of the proportion of individuals who survived the best isolates in suppressing individuals were EI.AB 2.1. This isolate can lower the individual to survive so that it can only develop until the instar 2 of nymph and nothing survives to become an imago. Based on the Lx value, it can be seen that the individual pattern of surviving from B. tabaci can be described in the form of a life span in Figure 1.

The proportion of individuals surviving B. tabaci on tomato plants introduced by isolates EI.AB.2.1 immediately decreased dramatically at 0.1 values on 12th day, Isolate KLE 3.3, SNE 2.2 and EI.AB 1.2 on the chart looks down dramatically on the 15th day, while isolates of EPL 1.1.4, EPL 1.1.3, TLE 1.1, TLE 2.2, negative controls and controls have almost similar chart, namely a gradual decrease in the pattern of sustained individuals and there are still individuals surviving live to become an imago.

DISCUSSION

The introduction of IEB in tomato plants can increase the resistance of tomato plants to B. tabaci. Tomato plants introduced with IEB can suppress B. tabaci egg laying and reduce the number of surviving individuals. Tomatoes given IEB showed fewer eggs than controls and controls sprayed with insecticides. The administration of IEB isolates can also reduce the amount of B. tabaci nymphs that develop in tomato plants, the number of individuals who succeeded in becoming imago in plants introduced with IEB was less than the control. EI.AB.2.1 isolate is the best isolate in suppressing the amount of B. tabaci individuals. This isolate showed B. tabaci, which survived only until the instar two nymphs did not develop into an imago. Besides EI.AB 2.1 There are isolates of KLE 3.3, SNE 2.2, and EI.AB 1.2, which show significantly different effects from controls but do not affect substantially the control sprayed with insecticides.

IEB can influence the number of B. tabaci nymphs that develop; this is alleged because the giving of IEB isolates will activate plant resistance signals that will affect the response of plants so that it can suppress the development of B. tabaci. According to Kloepper and Ryu (2006) states that non-pathogenic microorganisms will induce plant resistance by activating signals from jasmonic and ethylene acids which are then responded to by plants with the production of chemical compounds or cytological changes. Production of defense-related chemical compounds, such as flavonoids, lignin, and other secondary metabolites, which produce effective defenses against various plant pathogens and insect herbivores arranged in the pathways of JA/ET and SA (Valenzuela-Soto et al. 2010). IEB can suppress B. tabaci attacks on tomato plants; this is in line with Murphy et al. (2000) stating that administration of endophytic bacteria suppresses the number of white flea nymphs that act as TOMV virus vectors.

The introduction of IEB in tomato plants did not affect the length of the day required by B. tabaci for development per stage. The average time needed to develop per phase can be seen in Table 3. Days needed by B. tabaci to develop at each stage are almost the same, with a range of distance differences of approximately 1-2 days. This is because the resistance of plants induced by IEB does not affect the duration of B. tabaci development days. The length of the day required by B. tabaci is more influenced by other environmental factors such as temperature and type of plants. In line with this, Kurniawan (2007) reports that B. tabaci have a faster generation time in cucumber plants than chili plants.

Purbosari (2008), also showed that the imago B. tabaci life cycle at 29 Adeg C was more rapid than at room and 23 Adeg C temperature. B. tabaci on plants introduced by IEB isolates in the life table showed the highest individual mortality per development compared to controls, whereas controls showed that individuals survived the highest in each stage of development. EI.AB 2.1 isolate shows individuals surviving with a value of Lx 0.0 on the stage of development of instar three nymphs, pupae, and imago.

In the pattern of sustained individual velocities (Lx), it is seen that the life rate of B. tabaci, which was introduced by isolates EI.AB 2.1, KLE 3.3, SNE 2.2, and EI. 1.1 decreased sharply while in controls, insecticide control had a regular pattern and not until zero, there are still individuals who survive to become imago. B. tabaci could not survive on plants that had been induced by IEB, presumably because tomato plants introduced by IEB isolates could increase jasmonic acid signals in plants so that plants produced toxic secondary metabolites and inhibitor genes to suppress B. tabaci attacks. As revealed by Howe (2004), antagonistic bacteria produce compounds of salicylic acid, jasmonic acid, and ethylene, which influence the eating activity of pest insects by providing chemicals that act as secondary metabolites.

This is in line with the study of Valenzuela-Soto et al. (2010) that Bacillus subtilis on tomato plants will induce resistance to white flea insects by increasing the expression of JA-independent genes (including photosynthetic genes, phenylpropanoids, and terpenoid pathway biosynthetic genes) and genes JA-dependent genes include protease and proteinase inhibitor coding, thereby reducing the attack of white lice in tomatoes. Pineda et al. (2012) also reported that Arabidopsis induced by P. fluorescens WCS417r had increased resistance to the liquid-sucking aphid of the Myzus persicae plant, plants introduced by P. fluorescens showed stronger expression after the attack. The introduction of IEB isolates in plants will work indirectly, IEB will induce resistance by activating the jasmonic and ethylene acid signals and the production of secondary metabolites.

Secondary metabolite compounds produced by plants will affect the work of B.tabaci cells; this will cause physiological disorders that lead to the death of B.tabaci. Also, secondary metabolites can also reduce the birth rate, increase the birth of disabled individuals, and affect the sex of the individual produced. This was revealed by Chowa'nski et al. (2016) that secondary metabolite compounds produced by plants have a broad spectrum. Secondary metabolite compounds will disrupt the work of the cells so that it will disturb physiologically from insect and will cause death, disrupting the birth rate, causing the birth of individuals defects, affect the individual sex produced, reduce the number of individuals and affect the stage of development of insects.

Acknowledgments: Acknowledgments to Kemenritekdikti through the Unand Budget Implementation Entry List (DIPA UNAND) in accordance with the agreement letter on the assignment of Leading Applied Research - Cluster Research - Publications to Professors of Universitas Andalas (PTU - KRP2GB - Unand) with contract number: T/42/UN.16.17/PP.KP-KRP2GB/LPPM/2019.

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Publication:Journal of Animal and Plant Sciences
Date:Feb 12, 2020
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