Efficacy of Ageratina adenophora against White rust (Albugo candida): A laboratory and field study

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Jigme & Bajgai 2019, Bhutanese Journal of Agriculture 2(1) 26-34
Efficacy of Ageratina adenophora against White rust (Albugo candida): A laboratory and
field study
Jigmee, Ram Chandra Bajgaie
ABSTRACT
White Rust of Crucifers is caused by Albugo candida, which causes damage to quality and
quantity of the produce. Conventionally, fungicides are applied to reduce disease incidences;
however, improper and excessive use of chemical fungicides can be detrimental to the
environmental health. In this study, different concentrations (2.5%, 5%, 7.5% and 10%) of
freshly prepared aqueous extract of Ageratina adenophora were tested for antifungal activity in
vitro and in vivo against A. candida using poison-food technique and Colony Forming Unit
(CFU/ml). In vitro results revealed that 10% aqueous extract of A. adenophora was most
effective against mycelial growth and biomass formation of A. candida.10% aqueous extract
inhibited 92.7% of A. candida mycelial and total biomass formation followed by 78% at 7.5%
concentration. The lowest inhibition was observed in 2.5% concentration with 2.4%. The in vivo
antifungal activity of aqueous extract of A. adenophora was tested in the potted plants under
normal conditions. The result revealed that the preventive control is most effective than the
curative control. Preventive control of disease incidence using 10% aqueous extract led to
54.1% reduction, whereas the curative control reduced by mere 3.6%. Thus, 10% aqueous
extract of A. adenophora demonstrates the potential for the control and management of white
rust of crucifers.
Keywords: Albugo candida, Ageratina adenophora, Aqueous extract, Antifungal activity,
Curative control, Preventive control
1. Introduction
Agriculture is the key source of sustaining livelihood and enhancing human life (Pino, Sánchez&
Rojas, 2013). It supplies food and other essential commodities for human consumption. Apart
from fulfilling food requirement of a growing population, agriculture plays pivotal role in
improving the economy of a nation (Dutta, 2015). However, the damage and destruction inflicted
on the crops by various pest and pathogens such as insects, microbes (bacteria, fungi, viruses and
mycoplasmas), nematodes, weeds, animals and birds have posed serious challenges to farmers in
terms of sustaining food productivity (Yadav, Kewal & Choudhary, 2015; Koul, 2011).
White Rusts (also called as white blister) are caused by several species of Albugo, belonging to
the class Oomycetes. The disease attacks aerial parts of cruciferous plant including flower,
Corresponding author: ji gme011@gmail.com
eDepartment of Environment & Life Sciences, Sherubtse College, Kanglung, Royal University of Bhutan

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leaves and stem. It does not attack the root of a large number of cultivated and wild crucifer
plants (Sharma 2017). Cultivated plants which are susceptible to infection by white rust include
cauliflower, cabbage, radish turnip, broccoli, and mustard. White rust caused by Albugo candida
(Pers. ex. Lev.) Kunze (Saharan, Verma, Borhan & Singh, 2014) is an important and widespread
disease in the world. According to (Lahiri & Bhowmik 1993; Sharma, 2017), the pathogen
produces two types of infection i.e., local and systemic infection. Local infection is characterized
by the formation of raised creamy white sporangial pustules on the under surface of leaves and
on tender shoots whereas systemic infection is usually seen in young inflorescence and terminal
leaves. Conventionally, fungicides are applied to reduce the disease incidence of white rust;
however, improper and excessive use of chemically derived fungicides causes residual toxicity in
the non-targeted organisms and leads to environmental degradation. Sustainable crop production
needs eco-friendly methods of pest and diseases control. Therefore, the development and
synthesis of bio-pesticides could be one of the options in conventional crop disease control
system.
By nature, all plants synthesize and discharge numerous secondary metabolites, which enable
them to defend against pathogens, pests, animal attacks and harsh environmental conditions
(Cavoski, Caboni & Teodoro, 2011). According to Kumar, Singh, Sharma and Kishore (2017),
Ageratina adenophora produces numerous secondary metabolites, which have antimicrobial
(antibacterial and antifungal), antiseptic, analgesic, molluscicides and insecticidal potential.
Although A. adenophora has meritorious chemical contents of diverse medicinal and
antimicrobial properties, it is one of the most invasive weeds, where invasion of this weed has
replaced larger part of the vegetation coverage and thus considered as a major threat to native
biodiversity (Tripathi, Kushwaha & Yadav, 2006). Its allelopathic property makes it a noxious
weed and dominates over other species (Subba & Kandel, 2013).In spite of potentially helpful
biochemical characteristics and harmful biodynamic characteristic, the potential of A.
adenophora for controlling fungal diseases in crops has not been evaluated (Sobrinho, de
Morais, de Souza & dos santos Fontenelle, 2017). Thus, this study intended to evaluate the
efficacy of fresh aqueous extracts of A. adenophora against White Rust of Crucifers.
2. Materials and Methods
2.1. Plant material collection, plant extracts preparation and media (PDA) preparation
Fully developed aerial parts of A. adenophora were collected from Sherubtse College campus,
Kanglung, Trashigang, Bhutan. Aqueous extract of A. adenophora was prepared by grinding 20g
of fresh leaf materials in an electric blender by adding sterile distilled water at the rate of 10 ml/g
(Nashwa & Abo-Elyousr, 2012). The homogenates were filtered with Whatman No.1 filter
paper. Then the filtrates were centrifuged at 5000 rpm for 10-15 minutes at room temperature
and the supernatant were collected. The extracts were further diluted by adding sterile distilled
water to have ranges of concentration (2.5%, 5.0%, 7.5% and 10%) and stored in refrigerator at
4°C. Potato Dextrose Agar medium (PDA) was prepared by dissolving 100g of potato infusion,

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2.5g of dextrose and 10g of agar in 500ml of distilled water (pH 5.6±0.2). The dissolved medium
was autoclaved at 15lbs at 121°C for 15 minutes.
2.2. Isolation of Albugo candida, antifungal activity, colony forming unit (CFU) and biomass
evaluation
The infected plants were collected from local farmer in Kanglung gewog. The infected plant
parts were cut, packed in the polythene bag and brought to the lab. They were thoroughly washed
(leaf with shiny whitish pustules underside of Brassica juncea) in clean water followed by sterile
water (distilled water), and with the help of sharp sterile razor blade, the infected tissues along
with adjacent small unaffected tissue were cut into small pieces (25 mm squares). The cut pieces
were transferred into sterile petri dishes containing 1% of sodium hypochlorite for surface
sterilization for 30seconds. After surface sterilization, the sterilized pieces were transferred to
petri dishes containing PDA and incubated at 25°C for 72 hours. A portion of mycelium from
fungal colony was transferred to fresh potato dextrose broth for the pure culture.
The antifungal activity of aqueous extract of A. adenophora was assayed by poison-food
technique and further confirmed by calculating Colony Forming Unit (CFU). The plant extract
was incorporated into the molten PDB broth at a desired concentration at the ratio of4:1 (PDB
and Plant extract) and then mixed thoroughly with Vortex Shaker. Then the medium was poured
into 50 ml conical flask. The conical flasks were inoculated with 0.1 ml of fungal suspension and
incubated in the incubator at 25°C for 48 hours. After 48 hours, 0.1 ml of fungal suspension
serially diluted up to 104was transferred and cultured on the petri plates containing PDA using
spread plate method. The inoculated plates were incubated at 25°C for 48 hours. After 48 hours,
the colonies on the petri dishes were counted under digital colony counter. The inhibitory
activity of the extract was determined and evaluated using the following equation (1) modified
from John, Ragi, Sujana & Kumar (2014):
IAG = NFC –NFT
NFC × 100
Where, IAG = Inhibitory Activity of Growth, NFC = Number of Fungal colony in Control plate,
NFT = Number of Fungal colony in Treated plates
To extract fungal biomass, 5 ml of different concentration (2.5%, 5%, 7.5% & 10%) of leaf
extracts was incorporated into 20 ml potato dextrose agar broth in 50 ml conical flasks. The
flasks were inoculated with 0.1ml of fungal inoculums. The cultures were incubated for 8 days at
25°C and the fungal biomass was harvested through centrifugation at 5000 rpm for five minutes.
The fungal biomass pallets were collected and dried overnight in the oven at 35°C. The
inhibitory activity of extract against fungal biomass was calculated using the following equation
(2):
(1)

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IAB = DWC – DWT
DWC × 100
Where, IAB = Inhibitory Activity on the Biomass, DWC = Dry Weight of biomass in Control,
DWT = Dry Weight of biomass with extract Treatment
2.3. In vivo evaluation of extracts against Albugo candida
In vivo antifungal potential of A. adenophora aqueous extract was studied on potted plants under
normal conditions. The potted plants were divided into Control (C), Curative Control (CC) and
Preventive Control (PC). The plants in (C) and (CC) were infested with A. candida inoculum by
spraying, when the symptoms appeared, the plants in CC were treated with the extract. For the
preventive control test, the plants in PC were first treated with the extract by spraying and were
infested with A. candida inoculum after 24 hours as per John et al., (2014).
2.4. Disease assessment
Table 1.Scoring method for evaluating the efficacy of A. adenophora extracts (Modified from
Goss, Mafongoya, Gubba & Sam (2017))
Scale
Disease severity
0
No symptoms
1
Very few symptoms, 1-3 small lesions on one or two leaves
2
Small lesions on 3-5 leaves
3
Enlarged lesions on 3 or more leaves
4
Coalescing lesions forming wilted
5
Mildly chlorotic and appearance of green island as the leaf ages
6
Plants completely defoliated and dying
The disease severity index (DSI) was calculated by following equation (3) adopted from Alemu,
Lemessa, Wakjira & Berecha (2014):
DSI = σ[d × n
N × m ] × 100
Where DSI = Disease Severity Index, d = disease rating on each plant, n = number of plants in
each score, N = total number of plants examined and m = maximum disease rating possible.
The reduction of DSI on each plant was calculated using following equation (4):
PR = [ PVC –PVT
PVC ] × 100
Where, PR = Percent Reduction, PVC = Percentage Value of the Control and PVT = Percentage
Value of the Treatment group.
(2)
(3)
(4)

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3. Result and Discussion
3.1. Antifungal activity- inhibition percentage (IAF %) and Biomass of A. candida
Results revealed that all the extract concentrations (2.5%, 5%, 7.5% and 10%) showed positive
results in suppressing the growth of A. candida with variable potency. The growth inhibition
increased with increase in the extract concentration. The number of A. candida colony and
biomass formation was found to be inversely proportional to the extract concentrations. The
highest concentration (10%) of A. adenophora aqueous extract was found to be the most
effective inhibiting A. candida growth in the lab. Therefore, 10% aqueous extract was chosen for
experiments during the field test with the potted plants. Figure (1) and (2) depict the trends of
inhibitory activity and biomass formation of A. candida in aqueous extracts of A. adenophora.
Figure 1.Inhibitory activity of aqueous extract of A. adenophora against A. candida
Figure 2.A. candida biomass formation in the presence of different concentrations of aqueous
extracts A. adenophora.
The highest inhibitory activity of aqueous extract of A. adenophora against A.candida was
observed in 10% concentration with 92.7% inhibition followed by 7.5% with 78% inhibition.
The lowest inhibitory activity was observed in the lowest concentration (2.5%) with 2.4 percent
2.4
75.6
78
92.7
0
10
20
30
40
50
60
70
80
90
100
0.0% 2.5% 5.0% 7.5% 10.0%
Inhibition percentage (%)
Concentration of A. adenophora aqueous extract
0.154
0.069
0.04
0.029
0.012
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
Control (0%)
2.5%
5.0%
7.5%
10.0%
A. candida biomass in (g)
Concentrations of A. adenophora aqueous extract

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inhibition followed by 5% extract with 75.6% inhibition (Figure 1). There was a significant
difference in the IAF of 2.5% and 5% of aqueous extract. The increase in IAF with extract
concentration in the initial two concentrations was drastic. With one fold increase in extract
concentration, inhibition increased by more than 30 folds from 2.4% to 75.6%. This may be due
to increase in the metabolites concentration which is optimum for the inhibition. However, there
was not much difference between 5% and 7.5%, possibly because the dead cells fenced the
inhibitory chemicals to account for live cells. The other reason could be that the already dead
cells might have absorbed most of the inhibitory chemicals from the extract leaving the
probability of contact between live cells and inhibitory chemicals low (Zhang et al., 2013).
Therefore, even with the increase in extract concentration to 10%, the increase in inhibitory
effect is low (75.6% to 78%). Thus, IAF of aqueous extract of A. adenophora extract increased
with the increase in the extract concentrations. On biomass front, the plate showed numerous
colonies and massive growth of fungal mycelium in the control (0% extract), however, as the
concentration increased, the fungal biomass and number of colony decreased (Fig 2). Thus, A.
adenophora extract has a wide spectrum of fungistatic property against A. candida. The
fungistatic activity of A. adenophora against A. candida may be due to the presence of secondary
metabolites such as (mono-, sesqui-, di-, and tri-) terpenoids, phenylpropanoids, flavonoids,
coumarins, sterols, alkaloids (Zhang et al., 2013), flavonoids, chromens, lactones, flavones and
flavanones (Torres-Barajas et al., 2013). However, there was no attempt made to understand the
phytochemicals of aqueous extracts of A. adenophora responsible for such an activity.
3.2. In vivo evaluation of aqueous extract of A. adenophora against A. candida - Disease
Severity Index (DSI)
10% aqueous extract was selected for the in vivo test. The potted plants labelled as Control (C),
Curative Control (CC) and Preventive Control (PC) was used for the test. The test plants
(mustard green) infested with A. candida inoculums started to show symptoms after four weeks
of infestation. The symptoms included distortion of young leaves and flowers, swelling on the
stems and whitish lesions on the under surface of the leaves.
Table 2.Disease Severity Index (DSI) and % Reduction (PR) of disease incidence (A. candida vs
aqueous extract of A. adenophora)
Weeks
Disease Severity Index (DSI)
% Reduction (PR) of
Disease
Incidence
C(%
)
CC (%)
PC (%
)
CC (%) PC (%)
1
67.9
71.4
25.0
3.6 54.1
2
78.6
75.0
32.1
3
80.1
77.2
36.8
(C=Control, CC=Curative Control, PC=Preventive Control)

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After symptom development on C and CC, the test plants were further observed for three weeks.
In the first week, the highest DSI (71.4%) was recorded in CC test plants followed by control
plants (67.9%). The lowest DSI was recorded in the PC test plants (25%). In the second and third
week, the highest DSI was observed in the control. In the second week, the DSI were 78.6%,
75% and 32.1% for control, CC and PC respectively. Similarly, the DSI for the third week were
80.1% (Control), 77.2% (CC) and 36.8% (PC); from this it became evident that DSI recorded for
three weeks is highest in CC plant as compared to the plants in PC. It is apparent that the
infections are less severe in plants pre-treated with aqueous extracts of A. adenophora than those
attempted to cure of infection. It is because the pre-treatment of the plants with aqueous extract
of A. adenophora makes the environment around the plants unfavorable (toxic) for the
infestation by A. candida. It might have also boosted the hosts’ defense system by various
phytochemicals.
3.3. % Reduction (PR) of disease incidence
The percent reduction was calculated using equation (4). PC was able to reduce infection by
51.4% whereas the CC was able to reduce infection by only 3.6% as compared to control. From
this result, it is evident that the preventive control is much more effective than curative control to
prevent the A. candida infection using aqueous extract of A. adenophora.
4. Conclusion
The study confirms that the aqueous extract of A. adenophora has excellent antifungal activity
against A. candida. Thus, it demonstrates high potential in its use as alternative eco-friendly
agent in controlling, reducing and managing A. candida infection and incidences in crucifers. Its
antifungal activity is mainly due to the presence biochemicals such as alkaloids, flavonoids,
chromens, flavones, diterpenes, sesquiterpenic, triterpenes and flavanones. However, a clear
understanding of its bioactive phytochemicals vis-à-vis antimicrobial characteristic is essential so
as to further affirm and validate its potential at the chemical and physiological level, and to use it
as one of the important plant protection agents in integrated pest management (IPM) programs in
subsistence organic farming. Secondly, it is doubly advantageous, in that by using it as an agent
of plant disease control, the noxious weed population can also be effectively controlled.
Acknowledgement
Authors are grateful to the Department of Environment & Life Sciences, Sherubtse College,
Royal University of Bhutan for the laboratory facilities, and the reviewers for their valuable
comments.

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