Seed treatment using rhizobacteria as plant growth promotion of two chili variety (Capsicum annuum L.)

The research aims to find out the effect of pre-germination treatment seeds using rhizobacteria as plant growth promotion of two varieties of red chili peppers in the field. The experiment used a randomized design of factorial groups. Factors studied were varieties (V) and rhizobacteria types (R). The variety factor consists of 2 varieties namely PM999 (V1) and Kiyo F1 (V2). While the type of rhizobacteria factor tried consists of 8 treatment, namely, control (R0), Azotobacter sp. (R1), B. megaterium (R2), P. atmuta (R3), B. alvei (R4), Flavobacterium sp. (R5), B. coagulans (R6), B. firmus (R7) and B. pilymixa (Rs). Each treatment was repeated 3 times, so there were 48 experimental units. Each unit of experiment is represented by 5 sample plants. The data was analyzed using ANOVA and continued with DMRT test at real level α = 0.05. The results showed that vegetative growth and production of chili plants until the age of 45 days after planting in each variety is not dependent on the pre-germination treatment of seeds with rhizobacteria. But the varieties of chili plants used affect vegetative growth and production. PM999 varieties are superior to the Kiyo F1 variety. Pre-germination treatment of seeds using rhizobacteria is relatively effective in improving vegetative growth and yield of chili plants. Among the 8 isolates rizobacteria isolate Azotobacter sp., B. megaterium, B. coagulants, Flavobacterium sp., and P. atmuta relatively effective to provide an increased effect on the growth and production of chili plants.


Introduction
Chili peppers are one of the significant vegetable commodities in Indonesia. Statistical data on the area of chili cultivation in 2017 reached 120,275 ha, ranked first widest compared to other vegetable crops [1]. However, the productivity level still in the third ranks after potatoes and shallots. The average productivity of chili peppers has only reached 7.93 tons ha -1 , this figure showed the lowest when compared to its production potential that can reach 20 tons ha -1 [1].
Rhizobacteria symbiotic with plant roots, it also serves as plantigrowthipromotingirhizobacteriai (PGPR). The plant growth increased byiPGPR able to occur through one or more mechanisms associated with the physiological character of rhizobacteria and the conditions in rhizosphere environment. Indoliaceticiacid (IAA) is a functioning auxin chemical found in plants and act as pgpr toiincreaseitheigrowthiandiyield. The function of IAA for plants among others increases cell development, stimulates the formation of new roots, spurs the growth, stimulates the flower, and increases the enzyme activity [2]. Generally, plants are not able to produce enough IAA for their growth and development. Some strains of rhizobacteria able to synthesize IAA from precursors (base materials) contained in the root exudates as well as from organic matter. Depending on the concentration, this active compound can increase or inhibit the plant growth [3].

Materials and methods
Rhizobacteria isolates were isolated from the healthy rhizosphere of chili plants in Cikanyawar Village, Sirna Galih I and Sirna Galih II, Mega Mendung District, Bogor Regency, West Java Province, Indonesia. Total of 10 g of soil around the roots were taken from several plants, then mixed and stirred until homogeneous. Soil sample composite as much as 10igiwasisuspendediini90imliof sterile water, then diluted to 10 -5 , 10--6 , and 10 -7 dilution levels. 0.1imliofisoilisuspensioniwas poured and stirred into King's B medium which was added with cyclohexamidei50imgiL -1 ,iampicillini50img L -1 , andichloramphenicoli12.5imgiL -1 iforithe isolation of rhizobacteria P. fluorescens [8]. To isolate rhizobacteria from the group Serratia spp. 0.1imliofisoilisuspensioniwasipoured into King's B media with 100 mg L -1 tetracycline antibiotics and 500 mg L -1 ampicillin [9]. Isolation of Bacillus spp. This is done by heating the soil suspension to a temperature of 80 0 C for 30 minutes in a water bath and then pouring it into tryptic soy agar (TSA) media with a concentration of 0.1. The rhizobacteria culture obtained was incubated at 27 0 Cifori48ihours.iEachigrowingicolonyiwasiisolatediandipureiculture made. Rhizobacteria that potential as plant growth promoters were identified using a standardized test procedure by Schaad et al. (2001) method [8] and Holt et al. (1994) [10]." The experiment used a randomized design of factorialigroups. Research factors were varieties (V) and rhizobacteria species (R). The variety factor consists ofi2ivarietiesinamelyiPM999i(V1)iandiKiyo F1 (V2). While the species ofirhizobacteriaifactoritriediconsistsiofi8ilevels,inamely,icontroli(R0), Azotobacter sp.(R1), B. megateriumi(R2), P. atmutai(R3), B. alveii(R4), Flavobacterium sp.i(R5), B. coagulansi(R6), B. firmusi(R7) and B. pilymixai(R8).iEachitreatmentiwasirepeatedi3itimesisoithatiin total there were 48iexperimentaliunits. Each unit of experiment is represented by 5 sample plants. The data wasianalyzediusingiANOVA,iandicontinuediwithiDNMRT test at a real level α = 0.05." Before seedling the chilli seed are treated with rizobacteria. Rhizobacteria isolates were previously grown in TSA medium"(Azotobacter sp., and Bacillus sp.,) or King's B (Flavobacterium sp. and Pseudomonas spp.), then incubated until 48 hours. Colonies of rhizobacteria are suspended in sterile water until the population density of 10 9 cfu ml -1 [11] or absorbant values OD600=0.164 (Azotobacter sp., and Bacillus spp.), and OD600=0,192 (Flavobacterium sp. and Pseudomonas sp.) using spectrophotometer. The chili seeds are then soaked in rhizobacteria suspense for 24 hours, then germinated." Germination of seeds is carried out in a seeding pot with measure 10 x l0 cm."Seedlings areimoveditoitheifieldiafteri4iweeksiafteriplanting. The planting media usediisiaimixtureiofisoiliand manureiwithia ratioiofi1:1 (v/v). Beforeiplanting in the field, the soil is first processed three times. Along with soil processing, manure is giveni15itonsiha -1 . The experimental unit is an planting shed with a width of 1 m and a length of 3.5 m. The shed planting used mulch black silver plastic. The planting distance was 50 x 60 cm. Maintenance includes fertilization and control of pests and weeds. Fertilizer used is a complete fertilizer NPK (15:15:15). Fertilizer dissolved in water at a dose of 2 kg 100 L -1 water. Fertilization is done once every 2 weeks by watering at the base of the stem."The 3 parameters observed include the height and stem diameter at 30 and 45 days after planting (DAP),ithe numberiofibranchesiandiproductiveibranches observediat 30 andi45iDAP, theinumber ofiflowersiandi theinumberiofifruits observediat 60 andi90iDAP. The data was analyzed usingiANOVA, which followed by a different test between the treatment and DMRT (Duncan Multiple Range Test) at α = 0.05."

Vegetative plant growth
The results showed that"the vegetative growth of red chili plants from eachivarietiesigaveitheisame responseitoitheitreatmentiofiseeds using various isolates of rizobacteria in pre-germination of seeds. All the observedigrowth parameters indicated the significant differences in value of the two varieties tried due to differencesiinirhizobacteriaiisolates usediinitheipre-germinationitreatmentiof seeds. However, the results of Test F tested a single factor, the varieties of chili plants are significant different on the observed vegetation growth parameters based on the height change of plants at 30 and 45 DAP, as well as the stem diameter at 45 DAP. The F test resultsialsoishowedithatiaisingleifactoriof pre-germination treatment of seeds using rizobacteria hadia noticeable effect on vegetativeigrowth parameters observed based on the high change of plant at 30 and 45 DAP, stem diameter ages 30 and 45 DAP. Theiaverageiheightiofiplantsiati30iandi45iDAP on two varieties and for each pregermination treatment of seeds with different types of rhizobacteria presented in Table 1 Table 1 shows that the chili plants height at 30 DAP and 45 DAP significantly varieties of PM999 produces higher growth ofiseedlings than the Kiyo F1 variety. PM999 variety was superior than Kiyo F1 variety."The differenceiinirhizobacteriaiisolatesiusediin theipre-germinationitreatmentiofiseeds is followed by differences in the value of vegetative growth parameters of different chili plants. Seed treatment using various rhizobacteria isolates from the results of this study has not shown a significant increase in plant vegetation growth when compared to control.iHowever, it can be seen that there is an increase in the height growth of chili plants at 30 and 45 DAP iniseeditreatmentiusingirhizobacteria isolateiFlavobacteriumisp., P.muta,iB. coagulans,iandiAzotobacter sp. is higher than control." Theiincrease of plant height"wasithoughtitoibeicloselyirelateditoitheiroleiofitheseirhizobacteriaias PGPR.iTheiabilityiofirhizobacteriaitoitheiproductioniofiIAAianditheiabilityitoidissolveiphosphate previously.iTreatmentiof seeds withiPGPRirhizobacteriaiplaysianiimportantirole,iespeciallyibeneficial initheiprocess of seed germination [12]. ApplicationiofiPGPRienhancesidirectly phosphates  [19]. Three PGPR strains (Microbacteriumioleivorans, Brevibacteriumiiodinum, and Rhizobiumimassiliae) exhibitedisignificantly greater plantiheight, fresh weight,idryiweight, anditotal chlorophyllicontent thanicontrol treatment on chili plant [20]." The results of the diameter stems of chili plants at 30 and 45 DAP ( Table 2) showed that the pattern is almost the same as the results of measurement of plant height. In stem diameter observation, rhizobacteria isolate Flavobacterium sp., Azotobacter sp. B. firmus and B. pilymixa had a positive impact on the increase the diameter stem. Auxinitriggersiainumber of cellularifunction rangingifrom differentiationiof vascular tissues,iinitiation ofilateral and adventitiousiroots, stimulation oficell division, elongationiof stems andiroots, and orientationiof root and shoot growth in response to light and gravity [21,22]. Six rhizobacteria strains, were produce IAA that able to increased shoot, stem and root length of chilli pepper [23].

Generative plant growth
The results"showed the reproductive parametersiof the red chiliiplants of eachivariety tried to provide the same responseito the treatmentiof rhizobacteria isolates usediin the pre-germinationitreatment of seeds. The entire of theithe production parametersidid notishow significantidifferences in valueifrom the twoivarieties triedidue to differencesiinirhizobacteria isolatesiused in the pre-germination treatment of seeds. However,ithe single factor varieties significantly affect the production of plants based onithe components of reproductive parameters that is the number of fruits at 8-90 DAP. The F test results alsoishowedithatia singleifactor of seed pre-germinationitreatmentiusing rhizobacteria species had not significantieffect oniall observed plant reproduction parameters based on the change in the numberiofibranches periplant, theinumber of productiveibranches, flowers andifruits periplant ati30-90 DAP. Averageinumberiofibranches periplant, productiveibranches, flowersiandinumber ofifruits on each red chiliivariety seed treatmentiwithivarious species ofirhizobacteriaipresented in Tables 3 and 4." Table 3ishows that the treatment of pre-planted seeds using 8 isolates of rizobacteria has not provided"aniincrease inithe valueiof red chiliiplant reproduction parametersiobserved basedion the change in theinumber ofibranches and theinumber of productiveibranches at 30-45iDAP, the numberiof flowers and the numberiof fruits periplant at 60-90iDAP. However, relatively the treatment of seeds using rizobacteria provides an increase in the number ofiflowers per plantimore in Description:"The numbersifollowedibyitheisameiletteriinitheisameicolumn (lowercase)iand theisame linei(capital letter) areino different thanithose initest level 0.05 (DMRT)." "PGPR can invigorate plant development through an assortment of instruments, including the fixation of atmospheric nitrogen, solubilization of phosphate, and production of phytohormones (i.e, indole-3-acetic acid (IAA), gibberellin, cytokinin, and abscisic acid), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and exopolysaccharide (EPS) [24]. Some PGPR strains of Agrobacterium, Azospirillum, Bacillus, Pseudomonas, and Rhizobium, able to increase plant growth [25]. Rhizobacteria strains (Microbacterium oleivorans, Brevibacterium iodinum, and Rhizobium massiliae) exhibited significantly greater plant height, fresh weight, dry weight, number of productive branches, flowers, fruits and total chlorophyll content than control treatment on chili plant [20]. Two rhizobacterial strains Streptomyces sp. and. Bacillus sp. were advance the chili plant height, wider the plant canopy, number of branches and number of fruit [26]." In the Table 4. chili plants derived from seeds that get pre-germination treatment using rizobacteria produce more fruit per plant than control treatment. Although the statistic is not increase significantly,"in the numberiof fruitsiper plant,ithe number of rizobacteria that give a positiveieffect to the increase in the number of fruits per plant there are only 5 isolates, ie. B.imegaterium, Azotobacterisp., P.idimuta, B.ialvei, and B.ifirmus. Depend on Table 4. two varietiesiwere tried toisee the responseiof chiliiplants to theitreatment of seedsiusing rizobacteria, it turns out thatiboth varietiesigive different responsesito seed treatmentibased on the changeiin the numberiof fruitsiper plant. PM999 varieties provide an excellentiresponse to seeditreatment using rizobacteriaicompared to KiyoiF1 varieties. As an evidenced in the measurementiof the numberiof fruit changesiper plant,ithat the varietyiof PM999 isisignificantly higher thanithe KiyoiF1. The PM999ivariety producesian average numberiof fruits periplant (51.98), while the KiyoiF1 variety producesionly an average numberiof fruits periplant (42.62). " The positive effects of rizobacteria in particular that belong to the group of plant growth booster agents (PGPR) can"take place through the production of IAA growth regulators, dissolving phosphates and induction of systemic resistance of plants. Colonization of the root system by rizobacteria increases the growth and development of root systems, recientence to abiotic insecurity, absorption and utilization of nutrients more efficiently [12,27]. Two rhizobacterial strains Streptomyces sp. and. Bacillus sp. were advance the chili plant height, wider the plant canopy, and promoted the number of fruit [26]. Two strains of rhizobacterial increased plant height, number of leaves, earlier flower's phase, and fruit weight of chilli [28]. Two rhizobacteria isolates were able to increase shoot length, root length, number of fruit, and weight of chili fruit [29]. " Based on the influence of single factors of varieties tried,"from theiresults of this studyiit can be concludedithat different varieties produce different growth and production. PM999 varieties were superiorito the KiyoiF1 variety, inivegetative and generative phase. The differences in nature of excellence areithought toibe related to theigenetic character ofibothivarieties. Eachivariety has differentigenetic characters that can beiseen by phenotype characters. " The results of observations in the vegetative growth phase of plants, it was shown that not all of the 8 rhizobacteria isolates tested had the potential as"plant growth-promoting rhizobacteria, as evidenced by the various benchmarks observed. Although statistically no rhizobacteria species have shown maximum results. The same thing has also been proven previously in the germination phase and seedling growth phase, which was observed based on seedling height, numberiof leaves,iand dry weightibiomass ofiseedlings. In addition, stand on theiresults of the physiological characterization of the rhizobacterial species, has theiability toiproduce IAAigrowth regulators andithe ability to dissolve phosphate. These two abilities are rhizobacterial characteristics that must be possessed by rhizobacteria that act as PGPR [12]. " The study"concludedithat rhizobacteriaiused in the pre-germinationitreatment of seeds showedithe role as PGPR, in the vegetative phase and reproductiveiphase. Theoretically, the level of production of a plant is highly dependent on the conditions of its vegetativeigrowth. The role of rhizobacteria that promote plantigrowth in theivegetative growth phaseiin this studyiturned out to also play a role in the plant reproduction phase. The group of rhizobacteria that are effective in the reproductive phase is related to the ability of these rhizobacteria as PGPR. One of the characteristics of rhizobacteria that act as PGPR is their abilityito produceigrowth regulators (auxins,igibberellins, andicytokinins),initrogen fixationiand the abilityito dissolve phosphateiand induce systemiciresistance toidisease. As previously stated, these rhizobacteria have played a role as PGPR sinceithe process ofiseed germination, seedlingigrowth, vegetative growth, thus it is suspected that the positive impact will also affectithe reproductiveiphase. The abilityito fix nitrogen, solubilizeiphosphate, and produceigrowth hormones (gibberellins, auxins,iand cytokinins) hasibeen widely reportedias a mechanismifor rhizobacteriaiin their role as PGPR [30][31][32]. "