Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Biocidal potential of Three Plant Extracts on

Rhizopus stolonifer, Causal Organism of Irish
Potato (Solanum tuberosum L.) Tuber Rot
Chuku, E.C, *Njoku, A.A. and Nmom, F.W.
Department of Plant Science & Biotechnology, Rivers State University,
Nkpolu-Oroworukwo, Port Harcourt, Nigeria.

Abstract:- Research study was carried out to assess the I. INTRODUCTION

biocidal effect of aqueous extracts of Curcuma longa,
Zingiber officinale, Citrus limon peel and synthetic Irish Potato (Solanum tuberosum L.) is an annual
fungicide Mancozeb against Rhizopus stolonifer using swollen underground stem tuber crop belonging to the
the poisoned food technique on PDA. Various family Solanaceae. It is an important tuber crop ranked
concentrations (50, 75, and 100%) of extracts from the fourth most important crop in the world (Ugonna et al.,
rhizomes of C. longa, Z. officinale, the peel of C. limon 2013; FAO, 2014). Since introduction, the production of
and Mancozeb (0.002%) significantly inhibited the potato has increased by over 120% in the past decade in
mycelia growth of R. stolonifer after 3 days. Effects of Nigeria and it is still grossly below demand (FAO, 2014).
the synthetic fungicide (Mancozeb) comparative to the Despite Nigeria being the highest producer of potato in
plant extracts were also determined. Although the Africa and second highest in the world, Nigeria is not an
extracts showed varying degrees of antifungal efficacy, exporter of potato (ITC, 2016). This has been attributed to
100% concentration of Z. officinale (58.96%) proved to various constraints. According to Ugonna et al. (2013),
be more potent against R. stolonifer than the other plant some constraints which limit Irish potato production,
extracts but was lower and significantly different when processing and marketing in Nigeria include Poor diseases
compared with Mancozeb (73.31%) at (P≤0.05) after 3 and pests’ management, inadequate storage facilities and
days. Extracts of C. longa and C. limon peel showed a inadequate supply of good quality seeds
lower inhibition level ranging from 45.01% to 56.98%
and 9.57% to 18.73% respectively and were Chukwu et al., (2008) stated that the availability of
significantly different when compared with Mancozeb tuber crops has been limited because of the unavailability
at (P≤0.05). Inhibition of fungal growth increased with a of good storage facilities and techniques. High percentage
corresponding increase in extract concentration and losses have been reported due to the in-effectiveness of
days. The plant leaf extracts effectively inhibited the traditional storage techniques such as pit storage and barn-
mycelial growth of pathogen in vitro after 3 days. In vivo heaping of the tubers. Losses between 40-60% have been
study was carried out using spore suspensions of R. attributed to poor storage methods and pathogens like
stolonifer. Fresh, healthy and surface sterilized Irish Mucor racemosus., Alterneria alternata., Rhizopus
potato tubers were inoculated with 6.4 x 10 4 spores/ml stolonifer, Phythophthora infestans, Pythium myriothylium,
and treated with aqueous extracts of C. longa, Z. Botryodiplodia theobromae, Fusarium solani and
officinale and C. limon peel after 24 hours. The result Fusarium oxysporum (Chuku et al., 2005; Ubalua and
showed that all plant extracts had significant effect on Chukwu, 2008). The effect of these losses can be translated
disease severity in tubers inoculated with R. stolonifer. to mean a decrease in revenue and threat to reliance on the
However, 100% concentration of Z. officinale gave the crop as a major staple carbohydrate food crop in this
best rot reduction caused by R. stolonifer with severity present economic situation.
score of 0.33 but it was not significantly different at
(p<0.05) from mancozeb which had a severity score of The use of chemicals have aided in the management
0.67. However, they were significantly different at of diseases but numerous problems such as, non-
(p<0.05) from the inoculated control (3.33). There were biodegradation, high cost, chemical residue retention in the
variations in weight loss but no significant difference plant produce, development of resistance in target
was observed among the various treatment methods organism, phytotoxicity, hazard to man and his
adopted. environment and sometimes non-availability have rendered
chemicals either difficult to adopt or farmers to have
Keywords:- Biocides, plant extracts, Rhizopus stolonifer, rejected them, in addition to various cultural and religious
Irish potato Tuber Rot. reasons (Abimbola et al., 1993; Okigbo and Odurukwe,
2009). Hence, there is a pressing need to find alternatives to
these existing methods with lesser or no side effects,
readily available and less expensive (Khulbe and Sati,
2009).

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Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
In many parts of the world, scientists have discovered  Pathogenicity Test
the many possibilities in using plants and their extracts for Pathogenicity test was carried out according to the
treating plant diseases (Sofowora, 1984; Okigbo and Igwe, methods described by Chuku et al, (2005) and Nwachukwu
2007). These higher plants have shown amazing potency as and Osuji (2008). A mycelia disk (5mm) of the test fungus
sources for new drugs although it is still largely under (Rhizopus stolonifer) from pure culture was cultured for 14
utilized. (Ahmedulla and Nayar, 1994). These plants days in a sterilized Potato Dextrose Agar (PDA) broth
derivatives are being used in several forms such as; liquid, containing 1g of PDA mixed in 140ml Sterile Distilled
powder, mixtures, spice, ointments, etc., (Apata, 1979; Wee Water. The culture was filtered using No 1 Whatman filter
yeow Chin, 1992). Plant-based products are generally paper and transferred into a 50ml distilled water containing
affordable, readily available, non-phytotoxic and easily 10% glucose.
biodegradable. Moreover, they are ecofriendly and stand as
alternative to chemical fungicides as reported by different The mixture was properly agitated and sprayed on
scholars (Akhilesh et al., 2012; Okigbo and Omodamiro, fresh surfaced sterile uninfected potato tubers. The tubers
2006 and Okigbo and Igwe 2007). This study was aimed at were kept at room temperature (28±2°C) for 14 days to
evaluating the biocidal potentials of aqueous extracts of examine for symptoms of rot and thus prove that the test
Curcuma longa, Zingiber officinale, Citrus limon peel fungi were able to cause infection.
against Rhizopus stolonifer, the causal organism of Irish
potato tuber rot.  Preparation of Stock Solution of the Plant Extracts
15 grams of each plant powder was mixed in 100ml of
 Sample Collection Sterile Distilled Water, stirred vigorously and kept for 24
Irish potato (Solanum tuberosum) tubers with rot hours in other to obtain a stock solution. The extracts was
symptoms were procured from Rumuokoro market in Obio decanted, filtered through a No. 1 Whatman filter paper and
-Akpor Local Government Area, Rivers State, South-South stored in a refrigerator at 40C and used within 48 hours. The
Nigeria. Fresh and apparently healthy tubers were procured stock solution of the plant extracts were diluted to 50%,
from the same market. Rhizomes of Ginger (Zingiber 75% and 100% concentration respectively. 50% was got
officinale), Turmeric (Curcuma longa) and fruits of Lemon by diluting 50ml of the stock solution to 50ml of distilled
(Citrus limon) were procured from the Fruit Garden Market water; 75% was obtained by diluting 75ml of stock solution
in D/line area of Port Harcourt, Rivers State. Synthetic in 25ml of distilled water; whilst 100% was 100ml of stock
fungicide (Mancozeb) was obtained from Agricultural solution only and was used in the in vitro experiment.
Development Programme unit (Rivers ADP) of the Rivers
State Government.  In vitro Bioassay: Effect of Plant Extracts and
Mancozeb on Diametric growth of the Pathogen-
 Isolation of Fungal Pathogens from Irish Potato Rhizopus stolonifer
Tubers Thirty nine (39g) of PDA powder was dispensed into
Infected potato tubers were surface sterilized in 5% 1000ml of sterile distilled water in a conical flask and the
Sodium hypochlorite and then rinsed thrice in sterile mouth was covered with non-absorbent cotton wool and
distilled water (SDW) according to the method of (Ritchie, aluminum foil. This was autoclaved at a temperature of
1991). Approximately 2mm cubes were cut from the tissue 121°C and pressure of 15atm for 15 minutes. About 2ml of
at the junction between healthy and infected portion of the different concentration of the plant extracts was mixed
potato tubers using a sterilized scalpel. These sections were with 10 ml PDA and dispensed into the Petri dish, the
inoculated to Petri dishes containing sterilized Potato medium was uniformly mixed and allowed to cool and
Dextrose Agar (PDA) and incubated at 28±2ºC for 7 days solidify. For positive control, 2ml of 0.002% of fungicide
and then examined for the development of fungi growth. (Mancozeb), obtained by diluting 2gms of fungicide in
1000ml of distilled water was mixed with 10ml PDA
 Subculturing, Purification and Identification of Test poured into Petri dish and allowed to solidify.
Fungi Pathogens.
When growth was established, subcultures were Two intersecting lines were drawn at the bottom of the
prepared using inocula from the different fungi in the petri dish to determine the centre of the plate. A disc (5mm
mixed cultures in order to obtain pure culture. This was diameter) of a 3 - 4 day old culture of the fungal pathogen
done by transferring the hyphal tips from the colony edge (Rhizopus stolonifer) was placed at the centre of each plate
of the mixed cultures to fresh plates of Potato Dextrose amended with various concentrations of plant extracts and
Agar using flame sterilized inoculating needle. After sub- fungicide respectively. The Completely Randomized
0
culturing, the plates were incubated at 28±2 C for 7 days. Designed experiment had 3 replications per treatment. The
control experiment had no plant extract treatment.
The process was repeatedly done until pure cultures were
Inoculated plates were incubated at temperature of 28±2ºC.
obtained. The resulting pure cultures were used for
characterization and subsequent identification of the The diameter of the growth of the test fungi was measured
fungal isolates based on their characteristics, using daily for three days and then used to detect the fungal
toxicity levels of the extracts using the formula according
identification guides by (Barnett and Hunter, 2008).
to Chuku et al. (2005).
Stock cultures were maintained on agar slants in
0
McCartney bottles and stored at 4 C in the refrigerator.

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Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
% growth inhibition = (DC-DT) / (DC) x 100/1 Completely Randomized Design experiment was replicated
Where DC = is the farthest diametric distance of thrice. The infected tubers were kept for 5 weeks at a
0
pathogen in control plate temperature of 28±2 C in order to access the disease
DT = is the farthest diametric distance of pathogen severity.
colony in extract incorporated plates
The severity of the infection after 5 weeks was
 In vivo Test Using Plant Extracts.
assessed by visual observation and scoring was done
This was carried out according to the methods of
according to Nwachukwu and Osuji (2008) based on a 0 – 4
Nwauzoma et al., (2017). About three 5mm mycelia disk of
scale.
the pathogen (R. stolonifer) from a 4 days old pure culture 0-no infection
was grown for 14days in a PDA broth containing 1g of 1-slight infection
PDA mixed in 200 ml sterile distilled water. The culture
2-moderate infection (50% of tuber infected.)
was filtered using No. 1 Whatman filter paper and
3-severe infection (75% of tuber infected)
transferred into 300 ml distilled water containing 10 %
4-complete rot (100% infection).
glucose. The mixtures were properly agitated and 6.4 x104
(spores/ml) suspension was obtained using a Also, Weight loss was observed weekly for five (5)
haemocytometer. This was sprayed separately on fresh weeks and percentage weight loss was calculated using the
surface sterile uninfected Irish potato at 10 ml per tuber.
formula,
Plant extracts at various concentrations of 50%, 75% and
% Weight loss = (IW-FW/IW) x 100/1
100% was sprayed on the tubers 24 hours later, using a
IW= Weight before inoculation
sterile manual hand spray pump. 10ml of 0.002% FW = Final weight after 7, 14, 21, 28 and 35days
Mancozeb, (fungicide) was applied as positive control.
Also, inoculated and un-inoculated controls were kept. The

II. RESULT

Treatments Percentage Inhibition (%)

Day 1 Day 2 Day 3
a a
Control 0.00 0.0000 0.00a
h j
Mancozeb 66.23 69.99 73.31i
c e
Z. officinale 50% 22.46 39.41 45.01e
de f
Z. officinale 75% 29.96 42.35 47.81f
f h
Z. officinale 100% 38.67 47.07 58.96h
C. limon Peel 50% 13.77b 7.05b 9.57b
c c
C. limon Peel 75% 19.99 11.76 14.35c
d d
C. limon Peel 100% 27.49 16.47 18.73d
d g
C. longa 50% 27.49 44.71 45.013e
e h
C. longa 75% 33.71 48.24 48.21f
C. longa 100% 44.97g 51.17i 56.98g
N.B. Means within the same column with different super script ( a,b,c,d) are significantly different (P<0.05) according to DMRT
Table 1:- Effect of Plant Extracts and Mancozeb on Diametric Growth of R. stolonifer after 3 Days

The effect of plant extracts on diametric growth of the inhibition percentage of 16.47% and 47.07% respectively.
pathogen after 24 hours is presented in table 1. Mancozeb The 100% concentration of C. limon peel (18.73%) had the
as well as the plant extracts inhibited the growth of R. lowest percentage inhibition among the plant extracts and
stolonifer by 66.23%. However it was significantly was statistically different (p<0.05) from 100%
different at (p<0.05) from the plant extracts. Among the concentration Z. officinale with a percentage inhibition of
extracts, 100% concentration of C. longa recorded a 58.96% after 3 days. The percentage inhibition of 100%
percentage inhibition of 44.97% and it was significantly concentration of Z. officinale (58.96%) was higher than and
different at (P<0.05) from 100% concentrations of C. limon statistically different (p<0.05) from other plant extracts but
peel and Z. officinale with percentage inhibition of 27.49% was lower and statistically different (p<0.05) from
and 38.67% respectively after 24 hours. Also, Mancozeb Mancozeb (73.31%) after 3 days.
had the highest percentage inhibition of the growth of R.
stolonifer with a percentage inhibition of 69.99 after 48 Table 2 shows disease development and severity
hours. However it was significantly different (p<0.05) from under each treatment method adopted after 5 weeks. The
the plant extracts. Among the extracts, 100% concentration rate of rot caused by Rhizopus stolonifer was reduced by
of C. longa recorded a percentage inhibition of 51.17% and the botanicals and mancozeb at each treatment method
it was significantly different (P<0.05) from 100% used. The 100% concentrations of Z. officinale gave the
concentrations of C. limon peel and Z. officinale with best rot reduction caused by R. stolonifer with severity

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Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
score of 0.33 but it was not significantly different (p<0.05) respectively. However, they were better and significantly
from 100% concentration of C. longa, C. limon peel and different (p<0.05) from the inoculate control with a score of
mancozeb with severity scores of 0.67, 1.33 and 0.67 3.33.

Treatments Rot Development

Z. officinale 50% 3±0c
Z. officinale 75% 0.33±0.58a
Z. officinale 100% 0.33±0.58a
C. limon Peel 50% 1.67±0.58b
C. limon Peel 75% 1.33±0.58ab
C. limon Peel 100% 1.33±0.58 ab
C. longa 50% 1.67±1.15b
C. longa 75% 1.33±0.58ab
C. longa 100% 0.67±0.58ab
Mancozeb 0.67±0.58ab
Inoculated control 3.33±0.58c
Uninoculated control 0.67±0.58ab
P value <.0001*
Significant Difference Yes
N.B. Means within the same column with different super script (a,b,c,d) are significantly different (P<0.05) according to DMRT.
Table 2:- Effects of Plant Extracts and Mancozeb on Rot Development after 5 Weeks Incubation

Table 3 shows the percentage weight loss in Irish potato tubers inoculated with R. stolonifer recorded weekly for 5 weeks.
The 75% concentration of C. limon peel recorded the highest percentage weight loss 9.65%, 12.73%, 16.39% for weeks 1, 2 and 3
respectively while inoculated control 19.12% and 25.44% had the highest percentage weight loss for week 4 and 5 respectively. C.
longa at 100% concentration had a percentage weight loss of 4.64%, 6.25%, 8.64%, 9.47% and 10.51% in week 1, 2, 3, 4 and 5
respectively. Mancozeb had a percentage weight loss of 6.4%, 8.5%, 12%, 13.03% and 13.93% in week 1, 2, 3, 4 and 5
respectively. There was no statistical difference (P≤0.05) in percentage weight loss in all treatments and all weeks.

Treatment
Week 1 Week 2 Week 3 Week 4 Week 5
Z. officinale _50 4.72±3.58 7.07±5.66 11.55±10.57 13.35±12.87 15.57±15.77
Z. officinale _75 2.08±0.43 3.08±0.43 4.84±0.51 5.33±0.59 5.87±0.62
Z. officinale _100 5.54±5.22 7.44±6.91 10.85±9.82 11.87±10.93 13.11±12.03
C. limon Peel _50 4.53±3.21 5.68±3.63 7.63±4.01 9.57±4.76 8.54±4.22
C. limon Peel _75 9.65±7.9 12.73±10.57 16.39±12.67 18.88±15.84 21.54±19.19
C. limon Peel _100 3.73±2.01 5.19±2.59 7.57±3.29 8.16±3.54 8.76±3.8
C. longa _50 6.02±4.06 7.91±5.13 11.71±8.13 17.39±16.39 24.58±27.48
C. longa _75 4.97±2.95 6.72±3.68 10.13±5.32 13.55±5.84 16.39±8.48
C. longa _100 4.64±3 6.25±3.75 8.64±4.9 9.47±5.38 10.51±5.94
Mancozeb 6.4±4.07 8.5±5.65 12±8.21 13.03±9.21 13.93±10.27
Inoculated control 4.04±0.59 6.02±0.5 10.05±3.26 19.12±17.66 25.44±28.07
Uninoculated control 5.41±5.17 7.44±6.64 11.22±8.83 12.6±9.68 13.63±10.64
P Value 0.8019 0.8428 0.9169 0.9063 0.8592
Significant Difference NO NO NO NO NO
Table 3:- Effects of Plant Extracts and Mancozeb on Weight of R. stolonifer Inoculated Tubers after 5 Weeks Incubation

III. DISCUSSION Popoola, 2007; Mohammed et. al., 2017). Tuber losses of
up to 25% has been attributed to this organism (Owunbiko
Rhizopus stolonifer is an important fungal pathogen and Mbanaso, 2005).
of Irish potato in Nigeria. It has been reported to cause
extensive rot of Irish potato tubers in storage (Clark and The inhibition of R. stolonifer by extracts of Z.
Hoy 1994; Onuegbu, 2002; Muhammed et al., 2004; officinale, C. longa and C. limon peel may be attributed to
Oyewale, 2006; Ameinyo and Ataga, 2006; Salami and the presence of antimicrobial substances, tannins,

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Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
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flavonoids and saponin found in the extract. This result is in RECOMMENDATION
agreement with the findings of Giriraju and Yumus (2013),
who reported that Z. officinale extracts showed It is recommended that the use of extracts of Z.
antimicrobial activities against some plant pathogens. Also, officinale, C. longa and C. limon peel should be encouraged
various studies of Z. officinale showed that gingerol an as part of an integrated approach for the control of Irish
important root extract of the plant, and shagaols, the potato tuber rot caused by R. stolonifer. It is also
dehydrated form of gingerols significantly inhibited the recommended that further investigations should be done on
growth of some fungal and bacteria pathogens (Mahady et the chemical nature of the active principles of the plants;
al., 2003; Azu and Onyeagba, 2007; Chen et al., 2008, Ali further investigations can combine the plant extracts for
et al 2008; Jiang et al 2006). In same way, extracts of C. possible synergistic effect.
longa have shown antimicrobial activity against molds and
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