A Possibility of using Antagonistic Bacterial isolates in Controlling Fusarium Wilt of Chrysanth (Chrysanthemum sp.)

One problem faced by chrysant farmer in North sumatera, indonesia is to control fungal disease caused by Fusarium oxysproum. The aims of this study were to know antagonistic bacterial isolates to reduce fungal disease caused by F. oxysporum and to know the isolates ability in producing plant growth hormon such as indole acetic acid and in solubilizing phosphate. Eight bacterial strains, collection of laboratory of Microbiology, Universitas sumatera Utara were tested against F. oxysporum in dual culture assay. two bacterial isolates, sP1, closely related to Bacillus amyloliquefaciens strain MPA 1034 and sP7R, closely related to Pseudomonas versuta strain l10.10 showed to reduce fusarium wilt in chrysant along with production of indole acetic acid and phosphate solubilizing properties in vitro. Application of the two antagonistic bacterial isolates resulted in considerably decrease of disease intensity caused by F. oxysporum. The results showed that utilization of bacterial isolate to promote chrysant performance is promising in the next future.

Local farmers generally perform fertilization and control of pathogenic fungi in chrysanths by using synthetic fertilizers and fungicides. The use of synthetic fertilizers and fungicides may cause soil damage, fungal resistance, resurgence, health and environment problem. An alternative to suppress Fusarium wilt is to utilise a biological control agent. This study examined two bacterial isolates SP1 and SP7R to control Fusarium wilt and to increase chrysant growth performance.

Isolation of fungal pathogen from chrysanth
Fungal pathogens were isolated from diseased plants of relatively cold area of Brastagi, exhibiting Fusarium wilt symptoms. Plant leaves were removed, cleaned with running water, cut into 1 cm 2 size, immersed in 70% ethanol for 1 minute, rinsed in sterile distilled water for 1 minute, then drained on filter paper. After drying, the sample pieces were placed on a petridish containing potato dextrose agar(PDA) medium. The culture was incubated for three days at ambient temperature until the fungal mycelium grew. The fungal colony emerging from the plant part is purified to new PDA.

Identification of fungal pathogen
Fungal pathogens were identified by using slide culture method. Isolates were inoculated at four points of block of PDA agar blocks measuring 1 cm 2 on the object glass, covered with glass, and then sterile distilled water was dropped on filter paper to keep moisturity. The slide cultures were incubated for 3-4 days, then slide observed under microscope. Fungal identification was performed using standard identification book of Gilman (1975) and Pitt et al., (2009)

in vitro antagonisms of bacterial isolates against F.oxysporum causative agent of fusarium wild in chrysant
Antagonisms of bacterial isolates against F.oxysporum were observed using dual culture assay. Bacterial isolates of rhizospheric bacteria of Brassica rapa pekinensis of relatively cold area of Brastagi (SP7R, SP9R, SP11R), endophytic bacteria of Brassica rapa pekinensis (SP5E, SP4E), and aerobic bacteria from liquid waste of oil palm (SP1, SP14, SP15) of previous study were collections from Laboratory of Microbiology, Universitas Sumatera Utara. Mycelium of F. oxysporum was cut and inoculated in the center of a petri dish containing a medium of PDA + 3% yeast extract. A total of 10µL of bacterial suspension, each with concentration of ≈ 10 8 CFU.mL -1 , was inoculated into blank paper discs (Oxoid), arranged on two side of fungal culture with a distance of 1 cm. The dual culture plates were then incubated for 7 days at amibient temperature. Inhibitory activity is indicated by the formation of inhibitory zones around the bacterial colonies towards fungal culture (Suryanto et al., 2016). Antagonism activity was further characterized microscopically by observing the mycelium at inhibitory zone of dual culture plates, showing some abnormal hyphae characteristics (Lorito et al., 1992).

Screening of plant growth promoting properties
Bacterial isolates were subjected to screen for their plant growth promoting properties i.e. phosphate solubilization and production of exogenous auxinIAA. To determine phosphate solubilizing activity of isolates, Pikovskaya medium was used in this assay (Pikovskaya, 1948). The plates were inoculated with bacterial isolates and incubated for 7 days at ambient temperature. The formation of visible and clear halo zones around bacterial colonies confirmed the positive result of phosphate solubilization. To determine the production of exogenous IAA of isolates, colorimetry method was used in this assay. The assay was done as follows: three millilitres of bacterial suspension (≈ 10 8 CFU.mL -1 ) was inoculated into 27 mL Luria-Bertani broth medium, supplemented with L-tryptophan in 100 ml flask, incubated in ambient temperature with 100 rpm agitation. To observe the trend of IAA production in culture, sufficient volumes of broth were sampled for every 4 hrs, and centrifuged at 5500 rpm for 10 minutes. The resulting supernatants were transferred into sterile reaction tube, filled with Salkowski reagent(4:1, v/v).The mixture was incubated for 20 minutes and measured for its absorbance using a spectrophotometer at a wavelength of 530 nm compared to the standard curve (ppm).

In vivo antagonism of bacterial isolates against F. oxysporum
The assay was done in greenhouse. Ten millilitres of bacterial suspension (≈ 10 8 CFU.mL -1 ) was inoculated into 2 kilograms mixture of soil and compost in pot. A 10 mL of sterile distilled water without bacterial suspension were used as a control. After 7 days chrysanth seeds were planted in the pot. Inoculation of 10 mL per pot of F. oxysporum (H" 10 6 CFU.mL -1 ) was done after 7 days of seed planting. Observations were conducted until 40 days including disease intensities, number of yellowing and wilting leaves, plant heights, and total number of leaves. Treatments of chrysant in pot were:

Cell number counted
One gram of soil in treatment was taken and diluted in sterile distilled water to make 10 -7 to 10 -8 suspension. One ml of suspension was inoculated into plate count agar and incubated at 31°C for 24-48 hours. Cell number was counted as colony forming unit per gram soil (CFU.g -1 ).

Molecular identification of potential isolates using 16s rRNA gene
Freeze-thawed method was utilised to break bacterial cell. Bacterial suspension of SP1 and SP7R in microtube was frozen at -10°C and thawed at 90°C for 10 minutes. This was done for five times for cell-breaking efficiency. Cellbreaking solution containing DNA was used for the amplification of the 16S rRNA gene performed using PCR method with a specific primer of 63f (5'-CAG GCC TAA CAC ATG CAC-3) and 1387r (5'-GGG CGG WGT GTA CAA GGC-3 ') (Marchesi et al., 1998). The PCR wascarried out for 40 cycles in a Thermo Cycler (LabCycler gradient) with the initial denaturation at 94°C for 2 min, cyclic denaturation at 92°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, with a final extension of 5 min at 72°C. The PCR products were checked visually by using agarose gel electrophoresis. Purification and commercially sequencing was done to the PCR product. The sequenced data were checked by BLAST analysis.

ResUlts isolation and identification of F.oxysporum causative agent of fusarium wild in chrysant
Eight isolates P1, P2, P3, PD1C, PD2, PD3, PD4, and PDX of potential fungal pathogens were isolated from diseased chrysanths were.One strain, P3 was further identified as F.oxysporum based on morphological characters. This isolate d showed white mycelium with pinkish or purplish center of colonies. Microscopy observation confirmed that 'P3 showed to have microconidia, and an ovalshaped, elongated, and septate macroconidia (Fig.  1). The confirmation of strain P3 as F.oxysporum was also compared to characteristics described by Pitt et al., (2009) and Gilmann (1975).

Dual culture assay on antagonistic bacterial isolates against F.oxysporum
Four bacterial isolates SP1, SP5E, SP7R, and SP9R showed to inhibit F.oxysporum growth (Fig. 2). It showed that bacterial isolates varied in inhibiting fungal growth ( Table 1). The varying degree of antagonism among isolates were assumed due to differences in production of antimicrobe substancies such as extracellular hydrolytic enzymes chitinase and glucanase into the dual culture medium. Fungal cell walls are mainly composed of chitin (4-9% of dry weight), followed by other component of 1,3-glucans. These polymers are the main target of hydrolytic enzymes of antagonistic bacteria (Benhamou et al., 1990;Rajarathnam et al., 1998). Suryanto et al.(2011) also stated that effectiveness of antagonistic bacteria in inhibiting fungal pathogens was affected by composition of fungal cell walls, differences in growth rate between bacteria and fungi, and competing metabolites produced by bacteria to attack fungi.
Antagonistic activity of antagonistic bacteria against F. oxysporum was observed through microscopical examination of abnormal hyphae. The antagonistic activity of four bacterial isolates caused F.oxysporum hyphae to develop abnormal growth such as stunted, curved, distorted, lysed, and dwarf hyphae when compared to normal hyphae (Fig. 3). Inhibition of fungal growth by antagonistic bacteria was shown by the inhibition zone around fungal radial growth. The fungal mycelium did not increase in length hence the tip of the mycelium looks short and flat. The abnormalities of the hyphae were assumed to be caused by hydrolytic enzymes produced by antagonistic bacteria within inhibition zones. Meanwhile the results of the research Asnita et al., (2012) stated that the antagonistic activity of chitinolytic bacteria causes F.oxysporum hyphae to be damaged, lysed, distorted and curve. According

Plant growth promoting properties of antagonistic bacterial isolates
Four antagonistic bacteria were subjected to phosphate solubilization assay in agar medium supplemented with insoluble tricalcium phosphate on Pikovskaya medium. The four isolates showed different ability in solubilizing phosphate as shown in Table 2. The formation of halo zones around colonies indicated the synthesis of extracellular organic acids which are able to bind ion Ca 3+ bound into insoluble form of Ca 3 (PO 4 ) 2 and free the ion H 2 PO 4 , forming a more clear zone than surrounding zone with bound P element in agar medium (Awais et al., 2017). Another mechanism to solubilize bound P element is by producing extracellular phosphatase that enzymatically hydrolyze P from its bound compounds (Parhamfar et al., 2016). Both mechanism is triggered genetically in which may explain the different ability among bacteria in effectively utilizing phosphate in environment (Khan et al., 2009).
Two bacterial isolates SP1 and SP7R capable to solubilise phosphate were chosen and subjected to IAA production assay. It was shown that SP7R produced more IAA copared to that of SP1 in 24 hrs of incubation. Higher production of IAA demonstrated during late logarithmic and early stationary phase of bacterial growth. Ji et al. (2014) reported the yield of IAA reaching 24.6 ppm during stationary phase of ten bacterial strains used in their study. Limited nutrients and Journal of Pure and Applied Microbiology low pH contributed to transcription of certain genes involved in catalysis of potential intermediet compounds into IAA. Addition of L-tryptophan into medium may also enhance synthesis of IAA (Spaepen et al., 2007). Our result might be considered higher due to the nature of bacterial isolate used in this assay. SP7R sampled from rhizosphere of B.rapa pekinensis showed to have high IAA production in this study. Rhizospheric bacteria are known as potential IAA producers because of their interaction with plant roots. Plant roots secrete exudates such as L-tryptophan to bacteria, in order to enhance the root growth by incorporating exogenous IAA synthesized by rhizospheric bacteria into plant (Khalid et al., 2004;Rosenblueth et al., 2008).

In vivo antagonism of two selected antagonistic bacterial isolates against F.oxysporum
It was observed that treated chrysanth with antagonistic bacteria did not show any Fusarium wilt symptoms during 40 days of planting as compared to that of (+) control, chrysant infested with fungal conidia (Fig. 4). Overall profile of plant parameters in terms of plant height and number of leaves were also improved in general (Fig. 5  and 6). Other studies have reported the potential of antagonistic bacteria in treating F.oxysporum infection in several plants. Paenibacillus sp. 300 and Streptomyces sp. 358 were reported to reduce Fusarium wilt on cucumber plants (Singh et al., 1999). Other study reported the potential of three antagonists, Streptomyces setonii, Bacillus cereus, and Serratia marcescens to reduce Fusarium wilt symptoms in the stem of tomato plants by F.oxysporum (Ferraz et al., 2014). Other factor besides hydrolytic enzymes produced by antagonistic bacteria in reducing Fusarium wilt were siderophores, cyanide, and antibiotics (Cantelan et al., 1999). Those products may be evaluated in future study to gain deeper  Before After application application understanding of antagonistic mechanisms from our isolates. It showed that SP7R increased more plant performance rather than that of SP1. The ability of SP7R to improve plant growth seemed correlating with high production of IAA. Bacterial strains capable of producing IAA might accelerate the growth of plants (Bolero et al., 2007). Several IAA-producing bacteria such as Acinetobacter, Alcaligenes, Arthrobacter, Azospirillium, Azotobacter, Bacillus, Beijerinckia, Burkholderia, Enterobacter, Erwinia, Flavobacterium, and Rhizobium were reported (Esitken et al., 2010). IAA functions as essential signal molecule to regulate plant growth, such as accelerating root growth, increasing resistance against pathogens, with final outcome on overall plant growth promotion (Shaharoona et al., 2006). Chrysant growth might also be supported by phosphate solubilization ability from isolates to provide sufficient plant needs of nutrients (Thakuria et al., 2004).

Bacterial cell number in pot soil
Bacterial cell number after 40 days of planting reached ≈ 10 13 CFU.g -1 (Table 3). This could be assumed that bacterial cell of SP1 and SP7R survived in all treatment pots and tend to thrive in the soils. Hence, cell densities between BSP1-BSP7R (promoting groups) and BSP1FR-BSP7RFS (antagonistic groups) did not differ. The abundance of soil organic matters in soil might support growth of most soil bacteria until reaching adequate cell number to execute their phenotypic roles in nutrient cycling and niches.

Molecular identification of antagonistic bacterial isolates based on their 16s rRNA gene
Two bacterial isolates, SP1 and SP7R were choosen to be identified. Isolation of gene fragment, 16S rRNA, followed by amplification in PCR using primers 63f and 1387r for 40 (Pingping et al., 2007;Wu et al., 2014;Wu et al., 2016). The bacteria had ability to synthesize antifungal lipopeptides, volatile organic compounds, and cell wall degrading enzymes (Wu et al., 2014). In this study, newly isolated P.versuta of relatively cold area of Brastagi for the first time was reported as biocontrol agent or plant growth promoting bacteria. The species was described as novel species by See-too et al., (2017), isolated from Antarctic soil. The prospect on evaluating further characteristics of both isolates as biocontrol and plant growth promoting agents is still needed for field application and deeper understanding of their natures.

DisCUssiONs
Antagonistic bacteria can be used as an alternative to control plant diseases and to increase plant growth. The ability of antagonistic bacteria to inhibit the growth of plant pathogenic fungi is thought to be due to their ability to produce chitinase and glucanase. According to Adams (2004), Chitinase and glucanase are hydrolase enzymes that can hydrolyze chitin and β-glucan which are the main components of fungi cell walls. Hydrolysis of chitin and β-glucan in the walls of fungi cells can reduce the integrity of the walls of fungal cells so that they cannot infect plants. Chitinase and 1.3 glucanase are enzymes that are key to inhibiting the growth of fungi. Bacteria can increase plant growth by dissolving phosphate and producing IAA. Phosphorus (P) is one of the nutrients that are important for plant growth, but its availability is very low in the soil because most phosphorus is bound by calcium (Ca) and magnesium (Mg) in calcareous soils and by iron (Fe) and aluminum (Al) in acidic soils, so there is very little available in plants (Liu et al. 2015), therefore phosphate solubilizing bacteria are needed in increasing the availability of phosphorus (P) in plants.
The ability of phosphate dissolving bacteria is related to the enzyme phosphatase and organic acids synthesized by these microorganisms. The phosphatase enzyme can decide which phosphate is bound by organic compounds to be the form available to plants. The presence of organic acids can directly dissolve phosphate which is the result of changes in PO4 3anions by acid anions or the binding of Fe and Al ions which previously bind P elements so that they can release phosphates which are bound and can be absorbed by plants (Khan et al. 2009). Meanwhile, the ability of bacteria to produce auxin depends on the phase of bacterial growth and the production of auxin is usually maximal in the stationary phase (Ji et al.

2014)
The direct mechanism of bacteria in increasing plant growth includes producing growth hormones, auxin, cytokinin, gibberellins, inhibiting the production of ethylene hormones, solubilizing phosphate , and nitrogen fixation. Plant growth is increased indirectly by bacteria through its ability to produce antimicrobial pathogens, siderofors and enzymes that can suppress the growth of fungi that cause plant diseases (Esitken et al. 2010).

CONClUsiON
Eight bacterial strains, collection of Laboratory of Microbiology, Universitas Sumatera Utara were tested against F.oxysporum in dual culture assay. Two bacterial isolates SP1 and SP7R closely related to Bacillus amylolique-faciens strain MPA 1034 and to Pseudomonas versuta strain L10.10 reduced fusarium wilt in chrysant. They produced indole acetic acid and capable to solubilise phosphate in vitro. Application of the two antagonistic bacterial isolates resulted in considerably decreased of disease intensity caused by F.oxysporum and increased chrysant performance.

ACKNOWleDGMeNts
We would like to express our deep appreciation to Universitas Sumatera Utara for partly supporting this study.