Biology and the Statistic Demographic of Aphis glycines Matsumura ( Hemiptera : Aphididae ) on the Soybean with Plant Growth Promoting Rhizobacteria ( PGPR ) Treatment

Indonesia heavily relies on soybeans as a source of food, feed for livestrocks, or raw materials for industries. Indonesia’s soybean production has fluctuated in five years between 2013–2018. Although Indonesia’s soybean production has been reported to reach approximately 982,598 ton in 2018, it was still not sufficient for domestic needs which was predicted to reach 2.8 million tonnes per year (Pusat Pengkajian Perdagangan dalam Negeri, 2019). Production fluctuation of soybean is caused by several factors including damage caused by plant pest and disease. Aphis glycines (Hemiptera: Aphididae) is a major pest on soybeans. A. glycines damage plant directly and indirectly by sucking leaf and stem fluids and decreasing quality and quantity of soybean productions. In addition, A. glycines cause indirect damage by being vectors of Soybean Mosaic Virus (SMV) (Widariyanto et al., 2017). Soybean yield loss by A. glycines has been reported to reach 58% (Wang et al., 1994); thus, A. glycines populations must be managed to stay below economic thresholds. Intergated Pest Management (IPM) is a pest management method based on environmental knowledge and is declared to be the main pest management approach in Indonesia. Good agricultural practice in maintaining plants’ health is the main component in IPM, since healthy crops are more resistant to pest damage. Plant growth promoting rhizobacteria (PGPR) is a method to increase plant resistances, using material that consisted of rhizophere microorganisms with activities that benefit crops (Kafrawi et al., 2015). PGPR can induce plant resistances to suppress pest populations (Soesanto, 2008). Resistances is the plant’s ability to harm pest and withstand pest attacks. Induced resistances on vegetative tissues will disturb feeding process and pest’s lives. Disturbed feeding will then affect the growth, development, and reproduction of the pest. Therefore, plant resistance is a limiting factor in the development of pest populations (Hutasoit & Sitanggang, 2018). Latifah et al. (2018) reported lower Bemisia tabaci ABSTRACT


INTRODUCTION
Indonesia heavily relies on soybeans as a source of food, feed for livestrocks, or raw materials for industries. Indonesia's soybean production has fluctuated in five years between 2013-2018. Although Indonesia's soybean production has been reported to reach approximately 982,598 ton in 2018, it was still not sufficient for domestic needs which was predicted to reach 2.8 million tonnes per year (Pusat Pengkajian Perdagangan dalam Negeri, 2019). Production fluctuation of soybean is caused by several factors including damage caused by plant pest and disease.
Aphis glycines (Hemiptera: Aphididae) is a major pest on soybeans. A. glycines damage plant directly and indirectly by sucking leaf and stem fluids and decreasing quality and quantity of soybean productions. In addition, A. glycines cause indirect damage by being vectors of Soybean Mosaic Virus (SMV) (Widariyanto et al., 2017).
Soybean yield loss by A. glycines has been reported to reach 58% (Wang et al., 1994); thus, A. glycines populations must be managed to stay below economic thresholds. Intergated Pest Management (IPM) is a pest management method based on environmental knowledge and is declared to be the main pest management approach in Indonesia. Good agricultural practice in maintaining plants' health is the main component in IPM, since healthy crops are more resistant to pest damage. Plant growth promoting rhizobacteria (PGPR) is a method to increase plant resistances, using material that consisted of rhizophere microorganisms with activities that benefit crops (Kafrawi et al., 2015).
PGPR can induce plant resistances to suppress pest populations (Soesanto, 2008). Resistances is the plant's ability to harm pest and withstand pest attacks. Induced resistances on vegetative tissues will disturb feeding process and pest's lives. Disturbed feeding will then affect the growth, development, and reproduction of the pest. Therefore, plant resistance is a limiting factor in the development of pest populations (Hutasoit & Sitanggang, 2018). Latifah et al. (2018) reported lower Bemisia tabaci and leaf spot incidences on tomatoes treated with PGPR compared to the untreated control. Rhizobacteria, component in a PGPR, can affect the interactions between plants and insects. Rhizobacteria are able to increase nutrition uptakes in plants causing an increase of food quality for insects. This is an example of induced tolerance due to rhizobacteria (Rashid & Chung, 2017).
The effects of PGPR on the survivorship and fecundity of A. glycines can be evaluated using life tables. Life tables can provide information on natality, development, reproduction, and mortality of each individual in a population. This study aimed to observe the ability of a commercially available PGPR product, containing Bacillus polymyxa and Pseudomonas fluorescens, in suppressing Aphis glycines Matsumura (Hemiptera: Aphididae) populations.

MATERIALS AND METHODS
This study was done at the Department of Plant Protection, Faculty of Agriculture, IPB University, Dramaga between January-April 2013.

A. glycines Rearing
Soybean seeds, of Grobogan variety, were used for A. glycines rearing and planted in 15 30× 30 cm polybags filled with soil and compose (2:1) at rates of 4 kg/polybag, compound fertilizer (containing nitrogen, phosphate, and potassium) 16-16-16 at rates of 0.5 g/polybag. Each polybag contained 6 soybean seeds and was watered every day. Initial populations of A. glycines were obtained from soybean fields located in Megamendung, Bogor. A. glycines were infested onto two-weeks-old soybean plants and allowed to reproduce ( Figure 1). Soybeans were closed with plastic cylinders and cloth mesh were placed on top. A. glycines were allowed to reproduce until required numbers were reached.

Effect of PGPR on A. glycines Biology
All surviving A. glycines were observed everyday to check whether individuals were alive or dead, molted (indicated by exuviae), and a count of the number of nymphs was done. A. glycines life cycles were observed since 1 st instar nymphs were infested onto plants until they reach imagoes. A. glycines went through 4 nymph instars before reaching imago. Development between instars were indicated by the existing of exuviae. Pre-oviposition stages were assumed since individuals reached imagoes until the first offspring were produced. Imago life time was counted since individuals reached imagoes until mortality. Fecundity were obtained from the number of nymphs produced by each A. glycines during its lifetime. Observation data were organized into a biological life table of A. glycines. Observations included the length of the 1 st , 2 nd , 3 rd , and 4 th nymph instars, life cycle, pre-oviposition, life length, and fecundity on PGPR treated and untreated plants.

A. glycines Cohort Rearing
Treatments used in this experiment were a PGPR treated and untreated control with 65 replications for each treatment. As much as 240 soybean seeds, variety Grobogan, were wash using clean water and air-dried on sterile paper for 15 minutes. Half of the seed batch were treated using PGPR and the other half was untreated as a control. The PGPR used in this experiment was Rhizomax®. Its formulation was powdery and contained the active ingredients of B. polymyxa dan P. Fluorescens. PGPR suspension were made form 50 g of PGPR products mixed into 5 L of sterilized water. Soybeans seeds used for the treated treatment were immersed in the PGPR  Figure 1. Two-week-old soybean plants Aphis glycines rearing suspension for 15 minutes, while untreated seeds were immersed in sterile water. Seeds were than airdried for 15 minutes. Soybean seeds were planted in 60 30 × 30 cm polybags. As much as 150 mL of the PGPR suspension remains was watered onto PGPR treated soybean plants, while sterile water was treated on the untreated control. This same watering treatment was done when soybeans reached 2 weeks-old. Soybean seeds used to feed A. glycines imagoes and produce 1 st instar nymphs were planted in 25 200 mL plastic cups filled with 200 g of growing media consisting of soil and compose (2:1). Two soybean seeds were planted in each cup. Soybean plant that have reached 7 days old were covered with plastic cylinders with cloth meshes on their top as previously mentioned (Figure 2). The following day after plants were covered, two A. Glycines imagoes were placed on plants and newly produced 1 st instar nymphs were obtained the next day.
First A. glycines nymphs were infested on 3-weekold soybean shoots that have been treated or not treated with PGPR. Plants were covered with plastic cylinders with the top and bottom covered with cloth meshes and polybags were placed on top of black cardboard.

Life Tables and Demographic Statistics of A. glycines
Surviving individual were counted every day to obtain survivorship data (l x ) of A. glycines. Daily fecundity (m x ) were calculated from the average nymphs produce by each imago at every stage (x). Survivorship and daily fecundity were organized into curves and life tables were obtained. Life table of cohorts are the life tables that records the development of each cohort by recording the survivorship of individuals until mortality of all individuals (Begon et al., 2006). Life table parameters of 1 generation of A. glycine were divided into 2-week periods starting from week 1 until week 37 (Price, 1997;Wilson & Bossert, 1971). Insect's demographic statistics according to Zeng et al., (1983) are quantitative analytic parameters of insect populations regarding to its survivalship, fecundity, and population growth patterns. These parameters include:

Data Analysis
Data variances were processed using Microsoft Excel 2007 and analyzed using a two-sample t test at α = 5% using Minitab 16.

The Effect of PGPR Treatment on A. glycines Biology
Results showed that life cycles of A. glycines were significantly different between the two treatments. Life cycles of A. glycines were longer on plants treated with PGPR compared to ones reared on untreated plants. This may be explained by the results from a study by Hutasoit & Sitanggang (2018)  and time required to reach imago stages will directly delay individual to reproduce, which is an important factor for insects to successfully infest plants. Kozlowski (1992) stated that delays of individuals to reach reproduction stages will increase mortality before reproduction, length of reproduction stages, offspring numbers and longer generation length. Life time of A. glycines were not significantly different between treatments at all life stages, except 2 nd nymph instars (  Fahimi et al., (2013) demonstrated that PGPR application significantly affected 1 st and 3 rd instar, but not significantly affect 2 nd and 4 th instar A. gossypii.
Short life time will affect fecundity. Fecundity and the length of pre-oviposition of A. glycines were not significantly different between the two treatments ( Figure 3). Food affect growth, development, fertility, mortality, and fecundity of insects (Begon et al., 2006).
Survivorship and mortality of A. glycines were similar between the two treatments used in the experiments (Figure 4)   supply Fe/iron to plants, produce lytic enzymes, and antibiotic properties (Jing et al., 2007). This shows that PGPR are able to induce plant resistances and accelerate A. glycines mortality.
A. glycines survival type can be categorized as type I. Many nymphs were produced daily based on the fecundity curves (m x ) ( Figure 5). In some occasions, there were A. glycines nymphs that were reared on both treatmetns that were able to produce offsprings. Food availability is an extrinsic factor that affect developmental time and reproduction of insects. Fecundity curve (m x ) continually increased after A. glycines individuals reach imagoes stage.
The highest fecundity reached 8.5 nymphs in one day was from untreated soybean plants and eight nymphs on PGPR treated soybean plants. These daily fecundities occurred peak several times on populations reared on untreated soybean plant, while from populations reared on PGPR treated plants only occurred once. This demonstrated that PGPR treatments were able to reduce the occurrences and level of maximum daily fecundity of A. glycines.

The Effect of PGPR on A. glycines Demographic Statistics
The value of A. glycines GRR on untreated soybeans was larger than ones treated with PGPR, reaching 104.861 and 71.834 individual/generation respectively ( Table 2). The number of female individuals produced by female imagoes (Ro) increased on untreated plants. Ro values from populations reared on untreated plants imply that the next A. glycines generation will increased by 63.326 folds from the previous generation and by 57.780 fold on PGPR treated plants. High GRR and Ro values indicate suitability of host for insects (Hidayat et al., 2019).  population growth for a certain period and compare reaction of populations to temperature, humidity, nutrient levels, or secondary metabolites from plants (Hutasoit & Sitanggang, 2018;Havlickova, 1987).
High rm values indicate that populations may continuously increase (Gill et al., 1989).
Doubling time of A. glycines reared on untreated plants was 1.184 days and 1.245 days from populations reared on PGPR treated plants. Low DT values may increase GRR and Ro (Efendi et al., 2018). The value of rm and DT can explain population growth in constant environmental condition and unlimited resources (Price, 1997;Southwood & Henderson, 2000).
PGPR is a soil rhizosphere microbes, which may increase plant growth and its resistances against pest and disease. Application of PGPR will directly affect plants by increasing the availability and mobilization or facilitate nutrients absorption, synthesize and alternate concentrations of various phytohormones that induce growth, and indirectly affect plants by suppressing activities of pest and disease by producing compounds and metabolites, such as antibiotics and siderophore, and systemically induce plant resistances (Zainudin et al., 2014;Walida et al., 2018).
The PGPR bacterial group Bacillus sp. and Pseudomonas sp. are the most studied genus due to their potential as biocontrol agents (Manik et al., 2018). Antibiosis is a suppressing mechanism used by the Bacillus sp. and Pseudomonas sp. Antibiosis is a PGPR mediated resistance mechanism against insect on plants that produces allelochemicals, such as chitinase enzymes, hydrogen cyanide (HCN), and siderophore. These allelochemicals have been reported to suppress reproduction, modify physiology, delay matureness, and induce physical behavior or abnormality on insects that eventually inhibit abundances of insect pest (Tuhuteru et al., 2019;Disi et al., 2019).

CONCLUSION
PGPR containing B. polymyxa and P. fluorescens were able to suppress A. glycines populations. Applications of PGPR caused longer development of 2 nd instar nymphs and eventually causing longer life cycles. This caused populations to grow slower than the untreated control.