Int. J. Agri. Agri. R.

International Journal of Agronomy and Agricultural Research (IJAAR)

ISSN: 2223-7054 (Print) 2225-3610 (Online)
http://www.innspub.net
Vol. 8, No. 4, p. 22-34, 2016
RESEARCH PAPER OPEN ACCESS

Pseudomonas fluorescens as plant growth promoting Rhizo-

Bacteria and biological control agents for white rust disease in
chrysanthemum

Hanudin*, Kurniawan Budiarto, Budi Marwoto

Indonesian Ornamental Crops Research Institute (IOCRI), West Java, Indonesia

Article published on April 22, 2016
Key words: P. flourescens, PGPR, white rust Puccinia horiana, biological control, in vitro and in vivo testing.

Abstract
The use of plant growth promoting rhizobacteria (PGPR) to control disastrous diseases in many crops has been
considered important recently. The research was conducted to evaluate several bacterial strains to control white
rust in chrysanthemum. The research consisted of two chronological experiments, in vitro and in vivo testing of
bacterial isolate against the disease. 16 bacteria isolates were collected, purified and applied on the rust-infected
leaf. Three isolates showed more effective in suppressing white rust during in vitro testing and further
identification confirmed these strains, Pf Kr 2, Pf Smd 2 and Pf Ktl were grouped into P. flourescens. In vivo
testing of the Pf isolates also revealed consistent performances of these three Pf isolates in retarding the growth of
fungal Puccinia horiana and even more effective than Azotobacter sp. and Azospirilium sp. The production of
ethylene on the leaf was coincidence with the slower development and lower disease intensity on the treated
plants. Among the three strains, Pf Kr 2 showed stronger suppression to the disease. Further investigations are
needed to further elucidate the existence of specific interrelation between Pf strains and plant genotypes or
cultivars. Prior to a selection of good bacterial inoculants, it is recommended to select cultivars that benefit from
association with these bacteria.
* Corresponding Author: Hanudin  [email protected]

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Introduction limited information have been known as biological

White rust is the one of the most disastrous diseases control of the diseases, especially for white rust.
at upper part of chrysanthemum plants in most world
production centers. It is caused by a fungal pathogen The mechanism of PGPR in protecting the plant from
by Puccinia horiana (Henn), an obligate parasite, pathogen infections was through several ways
which hosted in limitedly 12 species including involving the physiological and biochemical
chrysanthemum. The fungus attacks the plant by hypereactions as responses to the given bio-signals.
penetrating the leaf directly through the cuticle by One indicator on the plant mechanism is the
enzymatic digestion and then colonizes the mesophyll production of ethylene associated with the plant
tissue both inter- and intracellularly (Bonde et al., response to wounding, pathogen attacks and other
2005). The intensity of incidence fluctuates stressed (Bleecker and Kende, 2000). Ethylene
depending on susceptibility of cultivars, temperature appears to be able to promote either disease
and air humidity. Warm temperature and high air resistance or susceptibility depending on the
humidity are favorable to meet a rapid development particular plant-pathogen combination. These
and spreading of the disease in susceptible cultivars mechanism inferred that ethylene is produced after
(Wojdyla, 2004). the signal is received and may be a stimulus for
defense response by regulating a wide range of
The use of chemicals and screening of resistance defense regulate genes, including those encoding
varieties are the most common methods in pathogenesis-related (PR) protein (Kim et al., 2015).
controlling the disease in commercial nurseries. Ethylene may also play a role in disease symptom
These costly practices were often applied regardless development in correlation with the timing of
the presence of the symptoms and intensity of the increased ethylene production and development of
diseases to ensure the marketable flower quality. In disease symptoms on the plant (Goto et al., 1980;
several countries like Indonesia, however, no active Elad, 1990; Boller, 1991; Porcel et al., 2014).
ingredient is found in commercial fungicide
registered specifically for white rust control (Yusuf et Several groups of bacteria, like P. flourescens (Pf),
al., 2014). The application of chemical is also Azotobacter and Azospirilium have been observed to
considered as the last effort in integrated pest have potential use for controlling important diseases
management with the wise and precise considerations in several crops. Certain strains of Pf are able to
for type of the pest, method, dosage/concentration, reduce destructive attacks of Verticilium dahlia in
interval and environmental friendly (Thornburn, eggplant (Soesanto, 2001), Sclerotium rolfsii in
2015). peanut (Soesanto, 2004) and Fusarium oxysporum
in shallot (Santoso et al., 2007), hot pepper (Maqqon
The use of biological agent has been reconsidered et al., 2006) and gladiol (Soesanto et al., 2008).
important in controlling diseases in many crops Considered as plant growth promoting rhizobacteria,
especially in the last few decades. The interest was these bacteria produced 2,4-diacetylflouroglucynol
encouraged as a part of responses to public concern (Phl or DAPG) and siderophore (Raaijmakers and
about hazards associated with chemical pesticides. Weller, 1998; Soesanto, 2000) inducing root colony
The recognition of plant growth promoting to protect the plant from soil borne pathogens
rhizobacteria (PGPR), a group of beneficial bacteria, (Soesanto and Rahayuniati, 2009).
as potentially useful for stimulating plant growth and
increasing crop yield has been successfully applied in Azotobacter spp. was also known as broad spectrum
several crops, such as potato, radish, sugar beet and antifungal agents which was protecting the plants
sweet potato (Farzana et al., 2009). In from fungal pathogens through HCN and sidephore
chrysanthemum, however, only few antagonists with productions. Synergistic interactions of these two

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with other metabolites might function as stress Puccinia horiana and (b) in vivo testing of isolated
factors including local and systemic host resistance PGPR to control white rust in chrysanthemum.
that led for the suppressions against Rhizoctonia
solani in cotton, guar and tomato (Chauhan et al., Isolation and in vitro screening of isolated Pf
2012), Cercospora in groundnut (Mali et al., 2011) Pf strains were collected from the soil at the
and F. oxysporum (Boshale et al., 2013). These free rhizosphere of certain bamboo species (local name
living bacteria were able to fix N2, produce vitamins are ‘Gombong’ and ‘Bitung’) and healthy
and growth substances including IAA, gibberellins chrysanthemum leaves in several areas in West Java,
and cytokines which enhanced root growth and aided Indonesia (Table 1). For about 10 g bamboo root and
in nutrient absorption (Mali and Bodhankar, 2009). chrysanthemum leaves were separately collected and
put into elenmeyer containing 100 ml sterile distilled
The genus Azospirilium composed of free-living, water. The water containing the bamboo root and
nitrogen-fixing bacteria that are found to be chrysanthemum leaves was put into rotary shaker
associated with the roots and rhizosphere of several with speed of 150 rpm for 30 min. The solution was
grasses including sugar-cane, maize, sorghum, and then diluted (10-1) by taking 1 ml solution into 9 ml
rice revealing a broad ecological distribution. They distilled water. The diluted solution was shaken for
can colonize, by adhesion, the root surface or the 150 for another 30 min. The 10-1 suspension was
intercellular spaces of the host plant roots. The diluted with the concentration of 10-2 up to 10-10, then
potential role of these PGPR is to promote plant inoculated at 0.1% Triptic Soybroth Agar (TSA)
growth by several mechanisms including nitrogen medium. All the cultured bacteria were incubated for
fixation (Bashan et al., 2004) and phytohormone 48 h under temperature of 29-30C. The growing
production, such as auxins, gibberellins, cytokinins, colonies were selected and grouped based on their
and nitric oxide as signals of plant growth promotion. morphological features. The selected single colony
Several studies revealed that Azospirilium is was isolated and then, reinoculated to get the pure
phylogenetically closer to Rhodospirillum and can colony for further testing.
grow in the presence of sucrose as sole carbon source
and is also better adapted to soil acidity, which offers The in vitro screening test of the bacteria isolates
the bacterium additional advantages for colonization against P. horiana was carried out based on the
of plant root tissue in acid environments (Baldani and modified method of Suhardi et al. (2011) with the
Baldani, 2005). Considering the potential use of bacteria isolates as the biocontrol agent. The source of
PGPR as biological agents against fungal diseases, the white rust was the infected chrysanthemum leaves.
research was conducted to evaluate the effectiveness The source of inoculums was the infected leaves. The
of Pf, Azotobacter and Azospirillum in controlling leaves were selected for the early stage of infection,
white rust disease in chrysanthemum. characterized by less than 5 yellow spots per leaf with
no rust pustule (Fig. 1a). The leaves were soaked into
Materials and methods the bacteria solution for about 10 min. The base of the
The research was conducted at the laboratory of leaf petiole was wrapped with cotton that was
bacteriology and crop protection glass houses of the, previously also dipped in bacteria solution. The leaf
Indonesian Ornamental Crops Research Institute was then put into plastic jar containing wet cotton to
(IOCRI) from January to December 2013. The preserve the humidity for the white rust and leaf
research comprised of 2 experiments; (a) Isolation of during the screening test (Fig. 1b) based on the
Pf and in vitro screening of isolated Pf against treatment design. The leaf was sprayed with bacteria
solution within 3 days - intervals.

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Table 1. Source and date of collection of Pf to be tested for in vitro screening against chrysanthemum white rust
(Puccinia horiana Henn).
Isolate Code Source of isolates Location Date of Collection
Pf Kr 1 Healthy Leaf (Chrysanthemum) Ciherang, Pacet, Cianjur 12 June 2013
Pf Kr 2 Healthy Leaf (Chrysanthemum) Ciherang, Pacet, Cianjur 12 June 2013
Pf Kr 3 Healthy Leaf (Chrysanthemum) Ciherang, Pacet, Cianjur 12 June 2013
Pf Kr 4 Healthy Leaf (Chrysanthemum) Ciherang, Pacet, Cianjur 12 June 2013
Pf Jl Rhizosphere (Gombong bamboo) Jambu Luwak, Ciawi, Bogor 14 May 2013
Pf Km Rhizosphere (Gombong bamboo) Kertamukti, Cipatat, Bandung Barat 14 May 2013
Pf Mm Rhizosphere (Gombong bamboo) Mandalawangi, Cipatat, Bandung Barat 14 May 2013
Pf Bd Rhizosphere (Gombong bamboo) Bedungan, Ciawi, Bogor 14 May 2013
Pf Mj Rhizosphere (Gombong bamboo) Babakan Jawa, Majalengka 14 May 2013
Pf Ktl Rhizosphere (Bitung bamboo) Katumlampa, Bogor Timur, Bogor 14 May 2013
Pf Tt Rhizosphere (Gombong bamboo) Titisan, Sukalarang, Sukabumi 14 May 2013
Pf Smd 1 Rhizosphere (Gombong bamboo) Cadas Pangeran, Sumedang 14 May 2013
Pf Smd 2 Rhizosphere (Gombong bamboo) Cadas Pangeran, Sumedang 14 May 2013
Pf Smd 3 Rhizosphere (Gombong bamboo) Cadas Pangeran, Sumedang 14 May 2013
Pf Tp Rhizosphere (Gombong bamboo) Kampung Tipar, Ciawi, Bogor 14 May 2013
Pf Cmk Rhizosphere (Gombong bamboo) Cimangkok, Sukalarang, Sukabumi 14 May 2013

The isolates that were able significantly to suppress

the white rust development were selected and
cultured on King’s B medium. Pf isolate 18 taken from
the laboratory of bacteriology was also included in
this stage for a comparison of the selected isolates.
After obtaining the pure culture of isolates, the
Fig. 1. (a) The selected chrysanthemum leaf with
morphological characterization and identification of
early stage of white rust infection used for the source
isolates were conducted based on Schaad et al. (2001)
of inoculums, (b) the leaf was then put into plastic jar
and Price (1999) methods. The biochemical
containing bacteria isolates for in vitro screening test.
identification was performed based on Cowan and
Steel (1974) method, the color of the colonies on
The observation on the white rust development was
King’s and Na media and the growth rate at warmer
conducted at 1, 3 and 7 days after applications. The
temperature of 34-37C.
conditions pustule was determined based on Suhardi
et al. (2011) and the increase of number of developed
In vivo testing of isolated bacteria against white rust
pustules was counted. The percentage of white rust
Preparation of host plants
suppression by antagonist bacteria compared to the
The tested chrysanthemum variety was ‘Swarna
control was measured using the formula of :
Kencana’ that was categorized as susceptible to white
rust (Yusuf et al., 2012). The rooted cuttings were
PS = (T - C) × 100%
collected from IOCRI seed production unit planted in
polybags with the density of 5 cuttings/polybag. The
Where :
media used was a mixture of top soil, bamboo humus
 PS = Percentage of Suppression
and manure (70:15:15 v/v) with additional fertilizers
 T = Degree of infection on the treated plants
of 200 kg/ha urea, 300 kg/ha SP-36 and 350 kg/ha
 C = Degree of infection of control plants (untreated)
KCl. The newly planted cuttings were then put inside

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the glasshouse conditions and maintained under Scale Damage Criteria

standard cultural practices. Long day condition was 2: Low, infection detected on lower plant
stimulated by providing additional light using 16 watt leaves and the intensity ranges 5-10%
LED lamps during the night (10.00 pm to 02.00 am) from total leaf area.
every night for 30 days. The lamps were arranged 2 x 3: Medium damage, infection detected on
2 m for the distance among lamp points and 1.5 m middle and lower plant leaves and the
high from the plants. Obamectin (Syngenta Co. Ltd) intensity ranges 10-20% from total leaf
with the dosage of 18.4 g/l was sprayed once a week area.
for prevention against insect attack and irrigated 4: Heavy damage, infection detected on
water was also given twice a week to maintain the upper, middle and lower plant leaves
optimum growth of the plants. and the intensity ranges 20-40% from
total leaf area.
Application of bacteria isolates 5: Very heavy damage, infection detected
The selected bacteria isolalates from in vitro testing on upper, middle and lower plant leaves
and isolates of Azotobacter sp. and Azospirillium sp. and the intensity was more 40% from
that were previously reported effective in controlling total leaf area.
fungal diseases by Indonesian Center for Agricultural
Biotechnology and Genetic Resources (ICABGR) were Where :
used for in vivo testing againts white rust. Before
 I = Intensity of white rust infection (%)
planted, rooted cuttings were dipped for 15 min in the
 v = Scale of the observed damage
10-10 cfu/ml bacterial solution. The bacterial solutions
 n = number of infected plants categorized in the
were also regularly sprayed into the plant every 7 days
same damage scale
up to 60 days after planting. The sprayed solution was
 Z = highest scale of the observed damage
arranged in 0.5% in concentration with the density of
 N = total number of observed plant samples
10-10 cfu/ml based on Taufiq et al. (2010). The
solutions of 0.05% Methyl Jasmonate (Hersanti,
Concentration of ethylene production on the leaf was
2004) and sterile water (control) were also sprayed
measured on 14 days after treatment application
with the same frequency and intervals as the bacterial
(DAA). The leaf samples were compositely collected
treatments for comparison of the effectiveness.
from the plants from 3 replications. Ethylene was
Observation of the white rust development was
known to be directly connected with the activity of the
conducted from 10% plant samples at 3, 5, 7, 9 and 11
antagonist bacteria inside the plants (Goodman et al.,
weeks after planting. The disease development was
1986; Timmusk, 2003). A HPLC based analysis with
determined using Suhardi (2009) criteria as
the adopted method from from Association of Official
presented on Table 2. The disease intensity was
Analytical Chemist (AOAC) Methods (1995) was
calcuated using the following formula:
carried out at ICABGR. The quantification of the
 (v x n)
Intensity (I) = (Z x N) x 100 % ethylene concentration was calculated using Taufiq et
al. (2010) based on the comparison of leaf area of leaf
samples and standard of 100 ppm ethylene-producing
Table 2. Scale and damage criteria of white rust
area.
(Puccinia horiana Henn) infection on chrysanthe-
mum (Suhardi, 2009).
The comparative advantage analysis of each
Scale Damage Criteria
treatment was conducted in every steps of the
0: Not infected (symptomless)
respected treatment being comprehensively applied.
1: Very low, infection detected only on
lower plant leaves and the intensity not These was scored based on the present and frequency
exceed than 5% from total leaf area. of the treatment advantage after comparing to the

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others. The higher the value of advantages was Results and discussion
representing the more frequent of the treatment In vitro screening test of the bacteria isolates against
advantage presented. The advantageous treatment white rust
was scored as 1 and the less was given the score of 0 Bacterial isolates performed differently in the
(Djatnika et al., 2012). The scoring criteria for the suppression against P. horiana during in vitro
basis of determination on presence and frequency of testing. These could be seen from the development of
treatment advantages were: pustule based on the increment of pustule numbers
a. Number of pustules on the treated plant at 7 DAA after 3 and 7 days and developmental stage of pustule
were low, the increase of number of pustule from after 7 days after bacterial application (Table 3). The
3 to 7 DAA were low and percentage of number of pustule ranged from 25 to 87 per leaf after
suppression of the treated bacteria on pustule 7 days and the higher number of pustule was
development was higher than control. observed on leaf sprayed with sterile water (control).
Most pustules on control treatment also reached
b. Disease incubation was longer on the treated more advance in developmental stages, that higher in
plants, intensity was low after 84 DAA and the number of broken pustules (spore release) than
ethylene production on the leaf was higher. the other treatments.

Table 3. Number and developmental stage of pustules after 1, 3 and 7 days after isolated bacterial application
under in vitro testing against white rust.
Number and increment percentage of numbers of Developmental stage of pustule
Degree of
pustule after 7 days isolates application
isolate
(Days after isolates application/DAA) (DAA)
suppression
Increment Increment
Isolate Code White spot White against white
percentage of percentage of
White with spot with rust
1*) 3*) number of 7*) number of
spot *) unbroken broken compared to
pustule after 3 pustule after 7
pustule*) pustule*) control
DAA (%) DAA (%)
Pf Kr 1 32 a 34 a 6.25 75 a 120.59 5c 20 a 50 b 13.79
Pf Kr 2 27 a 27 a 0 30 b 11.11 0d 23 a 7c 65.51
Pf Kr 3 17 b 29 a 70.59 57 b 96.55 0d 35 a 22 bc 34.48
Pf Kr 4 29 a 37 a 27.59 63 a 70.27 3c 10 b 50 b 27.59
Pf Jl 27 a 28 a 3.70 47 b 67.86 7c 10 b 30 b 45.98
Pf Km 16 b 17 b 6.25 72 a 323.53 12 b 30 a 30 b 17.24
Pf Mm 16 b 17 b 6.25 67 a 294.12 3c 14 b 50 b 22.99
Pf Bd 27 a 32 a 18.52 69 a 115.63 35 a 15 b 19 c 20.69
Pf Mj 5c 6b 20 47 b 683.33 10 b 27 a 10 c 45.98
Pf Ktl 17 b 17 b 0 23 c 35.29 12 b 5b 6c 73.56
Pf Tt 12 b 15 b 25 73 a 386.67 15 b 23 b 35 b 16.09
Pf Smd 1 17 b 27 a 58.82 37 b 37.04 10 b 7b 20 c 57.47
Pf Smd 2 15 b 15 b 0 25 c 66.67 2c 11 b 12 c 71.26
Pf Smd 3 12 b 17 b 41.67 39 b 129.41 9 bc 10 b 20 c 55.17
Pf Tp 19 b 23 ab 21.05 49 b 113.04 12 b 17 ab 20 c 43.68
Pf Cmk 17 b 29 a 70.59 53 b 82.76 13 b 10 b 30 b 39.08
Control 15 b 35 a 133.33 87 a 148.57 0d 7b 80 a -
CV (%) 7,2 7.9 6,9 7,5 10,1 9,2
Remarks: values within the same column followed by the same letter were not significant under Duncan’s
Multiple Range Test (DMRT), α = 5%.

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The suppression of white rust development by consisted of three layers mucopeptide. The content of
bacterial isolates was apparent at 3 days after might as much as 3-12 % from the total dry weight.
application. The degree of suppressions was varied Mucoopeptide is a chemical compound composed of
among the bacterial isolates. Three isolates namely, units of n-acetyl glucosamine (nag) and n-acetyl
Pf Kr 2, Pf Ktl and Pf Smd 2 showed significant muramic acid (nam) bound in the composition , 1-4
suppression on the number of white rust pustules. (Kaiser, 2014). Mucopeptide complex was often
The increment of pustule on the leaves was absent (0) referred to by the name of murein (Acharya, 2013).
after 3 days application. The performances of these
three bacteria isolates in suppressing the newly Table 4. Cell and colony features of Pf Kr 2, Pf Ktl
developed pustule were consistent up to 7 days and Pf Smd 2 based on morphology and biochemistry
application. The increase of pustule number was less characteristics and predicted of isolate group.
than 10 pustule per leaf and these was much lower Characteristics of selected
Cell feature isolated bacteria
than the other treatments that might reached more
Pf Kr2 Pf Ktl Pf Smd2
than 20 newly developed pustules after 7 days. Morphology
1. Cell shape Rod rod rod
2. Doom elevation Flat flat flat
The less number of newly developed pustules on the 3. Cell margin circled circled circled
leaf treated by Pf Kr 2, Pf Ktl and Pf Smd 2 was 4. Gram (KOH 3%) - - -

seemed connected with the slower developmental

Biochemistry
stage of pustule. The development of white rust 1. Color of colony when
cream cream cream
pustules under these three isolates were retarded as cultured at Na medium
2. Color of colony when florescent florescent florescent
viewed from the lower number of white spot with cultured at King’s medium green green green
broken pustule after 7 days isolates application (Table 3. Growth at temperature
+ + +
of 34-37C
3). The spore of fungal pathogen were released and
spread from the body through the broken pustules. P. P.
P.
Predicted isolate group fluoresce fluoresce
The broken pustule was also an indicator of the fluorescens
ns ns
maturity of the spore. When the environment
condition was conducive, the fungal spore might The three isolates had flourescens green colony under

germinate and infect the leaf of the susceptible King’s B medium (Table 4). The flourescens green

cultivar (Hanudin et al., 2015). colony was caused by "pyoverdins" an iron chelating
agent that was produced by a bacterium when grown

Three isolates of Pf Kr 2, Pf Ktl and Pf Smd 2 showed under lacked-iron medium. Following the method of

better suppression against white rust compared to Godfrey and Marshall (2002), the three isolated

control. The degree of suppressions of Pf Kr 2, Pf Ktl bacteria (Pf Kr 2, Pf Ktl and Pf Smd 2) were grouped

and Pf Smd 2 were measured 66.51%, 73.56% and as Psedomonas flourescens, based on the

71.26%, respectively, compared to the control. Based morphological and biochemical features of their cells

on these observations, three isolates Pf Kr 2, Pf Ktl and colonies.

and Pf Smd 2 were selected for further evaluation on

their effectiveness against white rust Puccinia In vivo testing of isolated bacteria against white rust

horiana under in vivo conditions. Puccinia horiana

In general, the symptom of disease arose when the

Identification of selected bacteria isolates interaction among three factors, the virulent

All selected bacteria isolates showed rod with elevated pathogen, susceptible host and the environment were

with flat cell shape, ledge/wavy and included as gram conducive for the pathogen to grow and developed

negative based on KOH test (Table 4). Stojšin et al. (Francl, 2001). The early appearance of white rust in

(2015) stated that gram negative cell had a ccell wall chrysanthemum was recognized as whitish-yellow
spot on the upper leaf surface. The spot was then
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developed into central spot and discoloration from The suppression of Pf Kr 2, Pf Ktl and Azotobacter sp.
white to dark brown. On the lower surface of the leaf, against white rust disease from 7 to 84 DAP were
the color of early stage pustule was initially pink. The observed more effective compared to other isolates
pustule was then enlarged, white turn into brown. and control (Fig. 3). These viewed from the disease
Rust pustules were actually a collection teliospore intensity less than 15.55%. The effectiveness of the
that might germinate to form basidiospora and isolates was also detected at the percentage of
infected the plant (Suhardi, 2009). suppression that was averaged more than 58.84%
from the control. The disease intensity on the plants
The effectiveness of Pf Kr 2, Pf Ktl, Pf Smd 2, treated by Pf Kr 2, Pf Ktl and Azotobacter sp. was
Azotobacter sp and Azospirilium sp. against white recorded 2.22, 15.55 and 13.33% at 84 DAP with the
rust under in vivo conditions were varied. Disease percent suppression of 94.12, 58.84 and 64.72%,
intensity ranged from 0 to 37.78% with incubation respectively.
period from 7.67 to 38.67 days after planting (DAP)
(Fig. 2 & 3). White rust showed delay in incubation
period when treated by the isolates. The longest
postponement was detected at chrysanthemum plants
treated by Pf Kr 2, that prolonged up to 38.67 DAP,
followed by Pf Ktl and Azospirilium with the periods
of 11.67 and 10.33 DAP, respectively.

Fig. 4. Percentage of suppression of isolates

treatment and control against white rust disease
Puccinia horiana in chrysanthemum at 84 DAP.

The ethylene concentration on chrysanthemum leaves

was seemed to be negatively related with the disease

Fig. 2. (Form left to right) Development of white intensity. Higher ethylene production was observed

rust Puccinia horiana attacks symptom on when the disease intensity was high. These

chrysanthemum leaves, from the early visible stage phenomenon was observed on the plants treated by Pf

characterized by whitish spot and turn into dark Kr 2, in that the disease intensity was the lowest (2.22

brown with broken pustules. %) at 84 DAP (Fig. 3). The ethylene concentration on
the plant leaf was the highest that was detected up to
723.935 ppm (Fig. 5).

Fig. 3. Development of white rust intensity under Fig. 5. Ethylene concentration on the leaf of
different isolates application treatment and control chrysanthemum under different isolate treatment and
on 7, 28, 56 and 84 days after planting (DAP). control at 84 DAP (ppm).

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Comparative advantages of each isolate 528.044–723.935 ppm on chrysanthemum leaf

The comparative advantage of each isolate tretament treated by certain bacteria isolates.
was determined by the following facts :
a. Certain bacteria isolates were able to supress white The analysis of comparative advantages of each
rust diseases more effective under in vitro screening. treatment was presented in Table 5. Treatment of
The number of pustule at 7 DAP was categorized as isolate Pf Ktl, Pf Smd 2 and Pf Kr 2 had higher total
few (23-30 spot/leaf) and the percentage of pustule number of comparative advantages form the rest of
increment from 1 and 3 to 7 DAA was low (11.11- treatment. Pf Kr 2 showed the presence of all
66.67%). The percentage of supression of bacteria advantage parameters and had the highest, following
isolate againts white rust disease was measured high Pf Ktl which did not affect the disease incubation
(> 65%) (Table 3). period during in vivo testing. Based on these analysis
of comparative advantages, three P. flourescens group
b. Period of incubation of white rust plants was isolates namely, Pf Kr 2, Pf Ktl, dan Pf Smd 2 were
delayed (up to 38.67 DAP) on chrysanthemum plants predicted to have capability to control white rust
treated by certain bacteria isolates under in vivo disease Puccinia horiana in chrysanthemum. The
testing. The disease intensity was recorded lower (< supressive mechanism of PGPR to the disease was
15.55%) at 84 DAP, thus the degree of supression of thorugh several ways, incuding (a) systemic
the isolates againts white rust Puccinia horiana was resistance induction to the plant, (b) production of
considered high (> 58%). Ethylene production, as siderophore, an iron chelat, made the ion unvailable
predicted was correlated with the disease intensity. for the pathogen, (c) secondary metabolite synthesis
The lower disease intensity reflected the positive such as enzymes or cyanide that acted as antifungal
reaction of the isolates in systematically protecting agent, degrading the cell wall and supressing the
the plant from white rust attacks. The plant produced growth of fungal pathogen, and (d) space and
higher ethylene as a response to the non-destructive nutrition competitive abilities againts the pathogen
infection of the bacteria inside the plant body. The (Beneduzi et al., 2012).
higher ethylene concentration was observed up to

Table 5. Comparative analysis of the presence of advantage parameter of isolate treatment againts white rust
disease on chrysasntemum under in vitro and in vivo testing.
Frequemcy of advantage of each treatment on the parameters
Percentage Degree of
Degree of
Number of Disease supression
Condito supression of Incubati
of increment intensit of the Total of
ns of isolates on
Treatment pustules on the y after isolates Ethyene comparat
pustule againts white period
at 7 DAP number of 84 DAP compared producti ive
on rust compared under in
under in pustule at 7 under to control on advantag
certain to control vivo
vitro DAP under in vivo under in es
stage under in vitro testing
testing in vitro testing vivo
testing
testing testing
Pf. Smd 2 1 1 1 1 0 0 0 0 4

Pf. Ktl 1 1 1 1 0 1 1 1 7

Pf. Kr 2 1 1 1 1 1 1 1 1 8
Azotobacter
0 0 0 0 0 1 1 0 2
sp.
Azospirilium
0 0 0 0 0 0 0 0 0
sp.
Jasmonate
0 0 0 0 0 0 0 1 1
acid
Control 0 0 0 0 0 0 0 0 0

Hanudin et al.
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Data on Table 7 indicated that Pf Ktl, Pf Smd 2 and Pf Acknowledgment

Kr 2 were able to supress white rust attacks in The authors thanked to Indonesian Agency for
chrysanthemum based on in vitro and in vivo testing. Agricultural Research and Development (IAARD),
Several reports informed that the action of Pf was through Center for Horticultural Resaerch and
related to the production of certain substances that Deveopment (ICHORD), Indonesian Ornamental
was toxic to the fungal pathogen. Santiago et al. Crops Research Institute (IOCRI) that financed, gave
(2015) reported that Pseudomonads were the biggest suggestions, criticisms in the planning and
bacterial group that had the capability in producing implementation of research. The authors also wish to
antibiotic and have been applied as biocontrol agents thank to the students of Faculty of Agriculture,
in many important crops. Pf stain A 506 was University of Lampung Zahara Diamond Arie, Astri
succesfully applied to control fire blight on apple Ambun Suri, and Candra Susiyanti; and to Muhidin,
(McManus and Jones, 1994), Gaeumannomyces Ade Sulaeman, Ridwan Daelani, Asep Samsudin, and
graminis var. tritici pada gandum (Thomashow and all those who helped and worked during the conduct
Weller, 1988), Ralstonia solanacearum on tomato of the research and report.
(Mulya, 1997), Plasmodiophora brassicae on chinese
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