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ICFCM 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 533 (2020) 012040 doi:10.1088/1755-1315/533/1/012040

Effect of plant extracts and chemical fungicide on viability

and percentage of seed-borne fungal infection on calliandra
(Calliandra callothyrsus) seed

T Suharti, Y M M A Nugraheni, E Suita and B Sumarni

Forest Tree Seed Technology Research and Development Institute, Jalan Pakuan
Ciheuleut PO BOX 105 Bogor, West Java, 16001, Indonesia

E-mail: [email protected]

Abstract. Calliandra (Calliandra callothyrsus) is one of the potential tree species that can be
used as bio-energy resources. Unfortunately, fungi are one of the factors that can infect and
decrease the viability of calliandra seeds. Some efforts are needed to improve the viability and
inhibit fungal infection through biological or chemical treatments. The study was conducted to
determine the effects of various plant extracts and chemical fungicide on the germination
capability, as well as the inhibition of fungal infection in calliandra seeds. Parameters observed
in this study were seed germination and fungi infection percentage. Seeds were treated by
immersing them in hot water (five minutes) followed by chemical fungicides (i.e., benomyl,
mancozeb) or plant extracts (i.e., ginger, onion, garlic, turmeric) for one hour. The study design
used was a completely randomized design. The results showed that all the treatments could
increase seed viability significantly. The treatment with immersion in onion, ginger and
mancozeb was more effective on seed germination ability (88.67%, 8.33%, 86%) and reduced
in fungal infections (11.33%, 5.33%, 0%). These treatments resulted in a significant difference
between seed germination compared with the hot water and turmeric treatments. The best
germination and reduction of fungal infection on calliandra seeds were showed on treatment by
onion and mancozeb, respectively. The results revealed that garlic, onion, turmeric have a
significant effect in inhibiting all fungal species compared to the hot water treatment. Ginger
and onion extracts were effective treatments for increasing seed germination and controlling
the fungal infection.

1. Introduction
Calliandra (Calliandra callothyrsus) is naturally found in Mexico and Central America, and in 1936
calliandra seeds were sent from Southern Guatemala to Java [1]. Calliandra is the best species
compared to other species because it can grow in various altitudes and types of soil and has high living
capability. This species has a natural symbiosis with fixation nitrogen bacteria (Rhizobium spp.) and
arbuscular mycorrhizal fungi [2]. The benefits of this plant are a potential source of renewable energy,
pulp, bee fodder, intercropping, fallow plants, erosion control, land rehabilitation [3], and animal feed
[4].
A seed is one of the success keys of the forest plant development program. However, one of the
obstacles in the supply of seeds is the infection of pathogenic fungi in both fresh and stored seeds [5].
In stored seeds, the predominant fungus is generally storage fungus. However, there are several types

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ICFCM 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 533 (2020) 012040 doi:10.1088/1755-1315/533/1/012040

of field fungus which is carried to the storage area and can survive due to the existence of a surviving
structure. Infection of seed-borne pathogenic fungi can cause germination failure, decrease in seed
longevity inside the storage, cause diseases in seeds and plants, and also be permanently settled in the
soil [6]. Therefore seeds are treated to reduce fungal contamination often before sowing; one of the
treatments is the use of chemical fungicides.
The active ingredients that are widely used to control fungal pathogens are benomyl and mancozeb.
Benomyl and mancozeb are applicable to seed treatment [7]. Based on mobility within the plant,
benomyl can be categorized as a systemic fungicide, while mancozeb as contact fungicide [8].
Benomyl and mancozeb are effective in inhibiting mycelium growth and sufficient to stimulate the
germination of conidia [9].
However, the use of chemical fungicides can have a negative effect on human health, animals, and
also for the environment. Therefore, a low-cost and environmentally friendly alternative that can
reduce pathogenic fungal infections is needed that can increase the germination and producing healthy
plants. One of the advantages of using plant extracts to control pathogens carried by seeds is its
cheapness [10], so reducing the costs. Plants that have the potential to be anti-fungal are ginger, onion,
garlic and turmeric [11]. The extract from these plants can inhibit the growth of Alternaria alternata,
A. solani, Phoma sp., Fusarium sp, and Aspergillus sp., and also can increase the germination of peas
[12].
The use of plant extracts such as ginger, onion, garlic and turmeric has been carried out in many
types of agricultural plants, but there is not much information on the application of ginger, onion,
garlic and turmeric on forestry plant seeds. Therefore research on the use of biological fungicides is
required and comparison with chemical fungicides is also needed for forest plant seeds. The purpose
of this study was to determine the effect of some plant extracts and chemical fungicide on the
germination ability and the inhibition of fungal infection to calliandra seeds.

2. Experimental Method
2.1. Study site
This research was carried out in the pests and diseases laboratory of the Forest Tree Seed Technology
Research and Development Center, Bogor, Indonesia (6°35'52.5"S 106°48'39.8"E).

2.2. Extract of onion, garlic preparation

Onion and garlic extracts were obtained by grilling the fresh tuber until smooth using mortar followed
by the addition of sterile aquades. The seeds were washed with sterile distilled water. Seed treatments
were done through the following steps: the seeds were soaked in hot water and left cold for 24 hours;
the seeds were soaked in hot water and left cold for 24 hours and then soaked in 10% onion extract
solution; the seeds were soaked in hot water and left cold for 24 hours and then soaked in 10% garlic
extract solution; the seeds were soaked in hot water and left cold for 24 hours and then soaked in 10%
ginger extract solution; the seeds were soaked in hot water and left cold for 24 hours and then soaked
in 0.1% benomyl, and the seeds were soaked in hot water and left cold for 24 hours and then immersed
in mancozeb 0.1%. The seeds are sown on moist paper media and then put into the germinator
chamber.

2.3. Observations
Observations were conducted once every two days for one month. Observation parameters included
germination and percentage of fungal infections. Isolation of seed-borne fungi was carried out by
taking the different fungus colonies that were on the surface of the seeds and then put in 9 cm diameter
petri dish filled with PDA and then incubated for seven days. Identification was made by using a
microscope preparat by placing one dose of fungi colony in preparat glass, and the observed fungus
using the microscope was identified by comparing the morphology of the fungus with the fungus in
the fungus identification book [13]. Percentage of fungal infections is calculated using the formula
[14]:

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ICFCM 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 533 (2020) 012040 doi:10.1088/1755-1315/533/1/012040

Percentage of fungal infections = the number of infected fungal seeds x 100 %

total number of seeds
Remarks: The number of infected fungal seeds is a number that represents a part of the fungus that is present on
the surface of the seed during germination (1 month)

2.4 Data analysis

The study design used a completely randomized design with six treatments; each treatment consisted
of three replications, with each replication consisting of as many as 50 units. The data were analyzed
using one-way ANOVA.

3. Result and Discussion

All treatments significantly affected the germination of C. callothyrsus compared to controls (Table
1). Soaking the seeds with hot water could break seed dormancy, so the seed viability is higher than
the control. Soaking the seeds in a solution of ginger, shallots, and mancozeb after soaked in hot water
and then being left cooled for 24 hours, will produce higher germination than just soaking the seeds in
hot water and left to cool for 24 hours. Therefore, these treatments can increase the viability of the
seeds compared to just being given hot water. These results were in line with the [15] study, which
showed that ginger and mancozeb could increase the germination of Lens arietinum (lentils) and
Lathyrus sativus.

Table 1. Calliandra callothyrsus seed germination at several treatments.

Treatment Germination (%)
Control 58.67c
Soaked in hot water and left until 24 hours 75.33ab
Soaked in hot water and left until 24 hours and then soaked in 74.67b
turmeric solution 10% for 1 hour
Soaked in hot water and left until 24 hours and then soaked in 89.33a
ginger solution 10% for 1 hour
Soaked in hot water and left until 24 hours and then soaked in 88.67a
onion solution 10% for 1 hour
Soaked in hot water and left until 24 hours and then soaked in 74.67b
garlic solution 10% for 1 hour
Soaked in hot water and left until 24 hours and then soaked in 86ab
mancozeb solution 0.1% for 1 hour
Soaked in hot water and left until 24 hours and then soaked in 78ab
benomyl solution 0.1% for 1 hour
Remarks: Numbers followed by different letters indicate a significant difference using the Duncan test at a 95%
confidence level.

Ginger and onion treatments can increase the calliandra germination by 10.66% and 10%.
According to [10], onion extract can increase the germination of wheat seeds by 3.67%, while ginger
extract increases the germination by 9%. Plant extracts' ability to increase seed germination is caused
by plant extract capability to suppress the fungi growth that can be found in seed embryos [16]. In
addition to that, onion is known to contain auxin, which plays a role in seed germination [17].
Soaking the seeds with turmeric extract does not cause an increase in seed viability; however, it
actually decreases the viability if compared with the hot water treatment. It is suspected that there is a
growth inhibitory compound contained in turmeric. Turmeric contains diarylheptanoids
(demethoxycurcumin, bisdemethoxycurcumin, curcumin), which act as anti-fungus [18]. However, it
is also known as growth inhibitors that can inhibit the germination and growth of several types of
plants [19].

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ICFCM 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 533 (2020) 012040 doi:10.1088/1755-1315/533/1/012040

Soaking the seeds with garlic extract did not increase seed germination and even lowered the
germination, presumably because the concentration that had been used was incorrect. [20] reported
that exudates produced by garlic roots at concentrations of 0.1 and 0.2 mL- could increase the
germination of lettuce seeds while at concentrations of 0.4 and 0.6 mL-, seed germination was
decreased.
The identification result showed that there are five seed-borne fungal species of calliandra at the
time of storage, including Aspergillus sp., Penicillium sp., Fusarium sp., Cylindrocladium sp. and
Botryodiplodia sp. (figure 1). The dominant type of fungus was Aspergillus sp. and Fusarium sp.
Conidiophores and conidia forms were one way to identify the genus of fungi. Based on [13], conidia
of Cylindrocladium sp. were hyaline, filiform, straight, 1-celled (figure 1a.), Pycnidia of
Botryodiplodia sp. were black, single, globose, conidia ovoid, dark and 2-celled at maturity (figure
1b.); conidia of Fusarium sp. were hyaline with macroconida several celled, typically canoe-shaped,
microconidia 1-celled, ovoid (figure 1c). Aspergillus sp. had a hyaline, straight, round vesicle shaped
conidiophore at the tip of the conidiophores, bearing phialides at the entire surface, conidia 1-celled,
globose/ovoid (figure 1d). Penicillium sp. had conidiophore, hyaline, straight, branched near the apex,
ending in a group of phialides, conidia green pale/hyaline, 1-celled, globose/ovoid (figure 1e.). [6]
reported that seed-borne fungal species of Dipterocarpaceae were Aspergillus sp., Penicillium sp.,
Fusarium sp., and Botryodiplodia sp.
Penicillium sp. and Aspergillus sp. are categorized as ubiquitous fungi on tree seeds, fruits, and
cones, that act as saprophytes or weak pathogen. They can affect the vigor and viability of seeds that
are stored under RH condition while B. theobromae, cause rots in a wide range of host and many
Fusarium sp., causing seedlings damping off [6].
Fusarium sp. is a field fungus, but can be carried by seeds and maturate at the storage even in
orthodox seeds [6]. [21] reported that Fusarium sp. is one of the fungi that can lessen diseases on
calliandra seedling that had rot symptom on stem base or root hypocotyls.

a b c

d e

Figure 1. Seed-borne fungal species of C. callothyrsus: Cylindrocladium sp. (a),

Botryodiplodia sp. (b), Fusarium sp. (c), Aspergillus sp. (d), Penicillium sp. (e).

The treatment of seeds soaked in hot water and left to 24 hours has resulted in the highest
percentage of fungal infections. It is because hot water can soften the seed coat. High moisture on the
seeds due to soaking causes a suitable condition for the growth and development of seed-borne fungi
(Table 2). However, further treatment using plant extracts and chemical fungicides prevents or inhibits
fungal growth and can even induce seed resistance to fungal infections.

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ICFCM 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 533 (2020) 012040 doi:10.1088/1755-1315/533/1/012040

High moisture on the seeds due to soaking causes a suitable condition for the growth and
development of seed-borne fungi. However, further treatment using plant extracts and chemical
fungicides prevents or inhibits fungal growth and can even induce seed resistance to fungal infections.
All the biological and chemical fungicides that were used significantly affected the infection of
Aspergillus sp. fungi compared to hot water treatment, and also effectively inhibited Fusarium sp.
Ginger, turmeric, onion and garlic are known to have secondary metabolites that act as anti-fungus.
Garlic, onion, turmeric has a significant effect in inhibiting all fungal species compared to the hot
water treatment. Onion and garlic extract significantly suppressed the growth of Fusarium sp.
compared to ginger extract. Along with [22] reported that the rate of inhibition of fungus growth by
ginger is lower than the onion. Based on [12], reported that ginger extract produced the most
significant percentage of seeds infected by fungus followed by onion extracts, garlic and turmeric.
Meanwhile, ginger contains zingerone, shogaol and gingerol, sesquiterpenoid β sesquiphellandrene,
bisabolene, farnesene and monoterpenoid (β phellandrene, cineol and citral) [23].
Table 2. Fungi infection percentage carried by calliandra seeds on several treatments after 1 month.
Treatment Fungi infection percentage (%)
Aspergillus Fusarium Penicillium Cylindrocladium Botryodiplodia Total
sp. sp. sp. sp. sp.
A 5.33b 4ab 1.33a 1.33a 1.33a 13.33
ab
a a a a a
B 12.67 18 0 0 0 22a
C 4b 3.33ab 0a 1.33a 0a 8.67ab
D 1.33b 9.33a 0a 0.67a 0a 11.33abc
E 4b 0.67b 0a 0a 0.67a 5.33ab
F 6b 0.67b 1.33a 0.67a 0a 8.67ab
G 0b 0b 0b 0b 0b 0b
H 1.33b 0.67b 0a 0.17a 0a 4.67ab
Remarks: Numbers followed by different letters indicate a significant difference
using the Duncan test at a 95% confidence level.
A : control.
B : soaked in hot water and left until 24 hours.
C : soaked in hot water and left until 24 hours then soaked in turmeric solution for 1 hour.
D : soaked in hot water and left until 24 hours then soaked in 10 % ginger solution for 1 hour.
E : soaked in hot water and left until 24 hours then soaked in 10 % onion solution for 1 hour.
F : soaked in hot water and left until 24 hours then soaked in10 % garlic solution for 1 hour.
G : soaked in hot water and left until 24 hours then soaked in 0.1 % mancozeb solution for 1 hour.
H : soaked in hot water and left until 24 hours then soaked in 0.1 % benomyl solution for 1 hour.

Soaking the seeds in turmeric solution can effectively suppress fungus growth compared to the hot
water treatment. Curcumin is one of the compounds of diarylheptanoids which has anti-fungal ability
to inhibit the growth of P. notatum and A. niger by damaging the sandalwood cell wall that caused cell
death [24]. Garlic and shallots contain alliin [25], allicin and thiosulfonates [26]. [27] reported that
ajoene, which is a derivative of allicin, has the anti-fungal ability by damaging cell walls stronger than
allicin.
The fungus growth inhibition mechanism worked due to toxins produced by plant extracts being
applied against pathogens [28] and host resistance induction to disease. [29] reported that the presence
of hydroxyl groups in phenols was associated with toxicity to microorganisms, namely because
hydroxylation increase led to toxicity increase. Ginger extract can induce the production of host
defense that compounds such as phytoalexin. It may increase in peroxidase enzymes [30].
Mancozeb treatment has a significant effect on eliminating fungus compared to hot water
treatment. Similar results were obtained from the [14] research, which showed that there was no
fungal colonization of the seeds added with mancozeb.

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ICFCM 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 533 (2020) 012040 doi:10.1088/1755-1315/533/1/012040

The benomyl mode of action is by inhibiting mitosis and cell division [31], while mancozeb
inhibits cell metabolism [32]. Furthermore, [9] reported that mancozeb was very effective in inhibiting
conidia germination compared to benomyl.
The use of chemical fungicides such as benomyl and mancozeb effectively suppresses fungal
growth; besides that, mancozeb also can increase seed germination. However, the use of chemical
compounds is harmful to health and also the environment. Because of that, the use of biological
pesticides can be used especially for preliminary treatment of seeds because the materials used were
cheap, easy to obtain, and only a small amount of plant extracts were needed. The choice of plants as
bio-fungicides should also pay attention to other roles than as anti-fungus, which can increase seed
germination, not vice versa, which reduces seed germination. Another thing that should be considered
in the use of bio-fungicides is the correct concentration, which, in addition to inhibiting growth, can
also increase seed viability.

4. Conclusions
The treatments to maintain seed germination and to reduce the percentage of fungal infections were
ginger and onion extracts, applied after soaking the seeds with hot water and left cold for 24 hours.
Ginger treatment could increase seed germination until 14% and reduce the percentage of fungal
infections until 10.67% compared to hot water treatment. Onion treatment could increase seed
germination until 13.34 % and reduce the percentage of fungal infections until 16.67%.

Acknowledgments
The authors would like to thank Forest Tree Seed Technology Research and Development Institute for
the facilities and research funding and Ms. Dina Agustina for her contribution to this work.

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