Evaluation of Antifungal Activities of Five Plant Extracts against Pseudoperenospora cubensis (Downy Mildew) in Muskmelon (Cucumis melo L.) ...

Evaluation of Antifungal Activities of Five Plant Extracts against Pseudoperenospora cubensis (Downy Mildew) in Muskmelon (Cucumis melo L.) ...
Annual Research & Review in Biology

                             31(2): 1-6, 2019; Article no.ARRB.38157
                             ISSN: 2347-565X, NLM ID: 101632869

         Evaluation of Antifungal Activities of Five Plant
         Extracts against Pseudoperenospora cubensis
       (Downy Mildew) in Muskmelon (Cucumis melo L.)
                                                      M. J. Falade1*, O. A. Borisade1 and M. Aluko1
    Department of Crop, Horticulture and Landscape Design, Ekiti State University, Ado-Ekiti, Nigeria.

                                                                                              Authors’ contributions

         This work was carried out in collaboration among all authors. Author MJF designed the study,
        performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript.
        Authors OAB and MA managed the analyses of the study. Author OAB managed the literature
                                          searches. All authors read and approved the final manuscript.

                                                                                                    Article Information

                                                                                     DOI: 10.9734/ARRB/2019/v31i230041
                                                             Galley Proof
                            (1) Dr. George Perry, Dean and Professor of Biology, University of Texas at San Antonio, USA.
                             (1) Prisila A. Mkenda, Department of Biosciences, Sokoine University of Agriculture, Tanzania.
   (2) ABA-Toumnou Lucie, Laboratory of Biological and Agronomical Sciences for Development, University of Bangui, Central
                                                                                                           African Republic.
                                              Complete Peer review History: http://www.sdiarticle3.com/review-history/38157

                                                                                       Received 20 November 2017
  Original Research Article                                                             Accepted 18 January 2018
                                                                                          Published 25 March 2019


 Laboratory study was conducted to evaluate the effect of leaf extracts of five indigenous plant on
 conidia germination, growth and sporulation of Pseudoperenospora cubensis causing downy
 mildew disease of muskmelon. Extracts of five plant; mexican sunflower (Tithonia diversifolia), bush
 banana (Uvaria chamae), salt and oil tree (Cleistopholis patens), goat weed (Ageratum conyzoides)
 and African eggplant (Solanum macrocarpon) at Four concentrations (15, 30, 45 and 60%) were
 tested against the growth, conidial germination and sporulation of Pseudoperenospora cubensis in
 Results show that all the plant extracts significantly inhibited conidia germination and radial growth
 compared to the control. The extracts had no significant (p≤0.05) effect on sporulation. The rate of
 inhibition of growth and conidia germination was concentration dependent being highest at 60% for
 the extracts. The extracts of Solanum macrocarpon was the most effective followed by Ageratum
 conyzoides, Cleistopholis patens and Uvaria chamea whileTithonia diversifolia caused the least
 inhibition of growth and conidia germination. At 15, 30, 45 and 60% concentrations growth of

*Corresponding author: E-mail: falademosesjimoh@yahoo.com;
E-mail: rufus.owoeye@eksu.edu.ng;
Falade et al.; ARRB, 31(2): 1-6, 2019; Article no.ARRB.38157

 Pseudoperenospora cubensis on PDA modified with Solanum macrocqrponwere 3.79, 3.65, 3.33
 and 2.87; and 4.25, 4.12, 3.92 and 3.89 for PDA modified with Tithonia diversifolia. Similarly,
 conidia germination percentages recorded at same concentration of extracts S. macrocarpon were
 87, 85, 70 and 62% while that of T. diversifolia were 91, 87, 84 and 72%. The study shows that the
 plant extracts has the potential for inhibition of the pathogen.

Keywords: Muskmelon; Pseudoperenospora cubensis; conidial germination; growth; sporulation.

1. INTRODUCTION                                                        The extracts of many plants have been reported
                                                                       to be toxic to many phytopathogenic fungi. The
Muskmelon (Cucumis melo L) is a cucubit widely                         efficacy in plant disease management varies with
grown in many tropical and subtropical regions of                      the concentration of active ingredients in the
the world and consumed for its nutritional                             plant extracts and the strain of the fungus [8].
qualities [1]. World output in 2013 was 29.4                           The antifungal effects of goat weed (Ageratum
million tons (t) [2] with India being the largest                      conyzoides) [9], mexican sunflower (Tithonia
producer producing 15.1 millon t. It contains 53                       diversifolia) [10], bush banana (Uvaria chamae)
kcal of energy, 13 g of carbohydrates, 1.4 g fibre,                    [11] african garden egg (S. macrocarpon) [12]
12 g of sugar, 1.3 g of protein, 3126 IU vitamin A,                    and salt and oil tree (C. patens) are well known
40.56 mg vitamin C, 531.96 mg potassium, 3,360                         but their use in the management of downy
mg of folate and 0.3 g of fat [3]. The fruit when                      mildew disease of muskmelon has not been
consumed help to suppress hypertension                                 exploited. Based on this, it is imperative to
because of the richness in potassium, improves                         evaluate the effectiveness of hot water extracts
vision due to high level of vitamin A that                             of these plants in the management of P.
strengthens the eye muscle. It also helps to                           cubensis, the pathogen causing downy mildew
regulate the sugar level, thus controlling                             disease of muskmelon.
diabetes. Besides, the fruit helps to booster body
immunity by stimulating the production of white                        2. MATERIALS AND METHODS
blood cells [3].                                        Galley Proof

                                                                       2.1 Collection of Plant Leaves                        and
Downy mildew of muskmelon is an important                                  Preparation of Extracts
fungal disease capable of causing 100% yield
loss when not controlled [4]. The pathogen
                                                                       Leaves of T. diversifolia, A. conyzoides, U.
affects all parts of the plant, reducing crop quality
                                                                       chamae, C. patiens and S. macrocarpon were
and quantity. It is an obligate parasite that needs
                                                                       collected from Ekiti State University Teaching
living muskmelon plant to grow and survive.
                                                                       and Research Farm, Ado-Ekiti and air-dried at
Symptoms of the disease are yellow to brown
                                                                       ambient temperature (24±2°C) for 14 -28 days.
lesions on the upper leaf surfaces. The infection
                                                                       The dried leaves were turned into powder using
begins as small light green spots that are not
                                                                       a blender (Okapi®, Mixer-Grinder), packaged into
water- soaked on the upper leaf surfaces but the
                                                                       sealable nylon and refrigerated at 4°C.
spots enlarge and later turn to yellow or brown
                                                                       Thereafter, 60, 45, 30 and 15 g of the powder of
lesions [5]. The disease is spread from plant to
                                                                       each plant were weighed into 250 ml standard
plant by air borne spores and infection is
                                                                       flask and 100 mL of distilled water at 70°C was
favoured by wet weather.
                                                                       poured into each flask [13]. The flasks were
The disease can be controlled effectively by the                       maintained at this temperature in hot water bath-
use of fungicides and crop rotation [6]. The use                       shaker for 30 minutes and thereafter the liquid
of synthetic fungicides like benomyl had proven                        extract was separated by vacuum filtration,
very effective but the increased awareness of                          poured into standard bottles and refrigerated at
environmental     side    effects of synthetic                         4°C for subsequent use as the stock solution.
pesticides, development of resistant strains of
pathogens and toxicity to non-target organisms                         2.2 Isolation       and      Morphological
have tilted attention on the development of                                Identification of P. cubensis
alternative method of pathogen control. One of
these is the use of plant extracts which are                           Muskmelon plants showing distinct symptoms of
considered cheap and compatible with the                               downy mildew disease were collected from fields
farming practices of the farmers [7].                                  at Ekiti State University Teaching and Research

Falade et al.; ARRB, 31(2): 1-6, 2019; Article no.ARRB.38157

farm, Ado –Ekiti, Nigeria. The leaves were cut                        2.4 Effect of Hot Water Extract on Growth
into pieces of about 1-2 cm and surface sterilized
by immersion in 0.2%NaOCl for two minutes.                            In order to evaluate the effect of the hot water
This was followed by two rinses in sterile distilled                  extracts on growth, standard PDA media
water and spraying with 70% isopropanol. The                          (control) and plant extract-modified PDA based
sterilized leaves were kept inside a laminar flow                     media were prepared as described previously.
cabinet for 20-30 minutes to dry. Five sterilized                     The plates were inoculated at the centre with 10
leaf cuttings were appressed unto the surface of                      µL of conidia suspension containing 1 x 102
Potato Dextrose Agar (PDA) (Sigma-Aldrich)                            conidia ml using micro-pipette (Eppendorf 1-10
containing 0.05% chloramphenicol (company                             µL).They were sealed with parafilm and
purchased) inside 9 cm sterile Petri dishes and                       incubated at 20°C for eight days. The treatments
removed. For the isolation of the downy mildew                        and the control were replicated three times. Daily
pathogen, three of the surface sterilized leaf                        measurement of the colony diameter along two
cuttings were placed on PDA containing                                orthogonal axes which were marked on the
chloramphenicol to prevent growth of bacteria                         plates commenced at 24 hours after inoculation
[14]. The plates were sealed with parafilm and                        and this continued for 5-10 days. The values of
incubated separately at ambient temperature for                       the growth rates were averaged and the
5-6 days. There was no growth on the plates                           percentage inhibition of mycelia growth (PIMG)
unto which leaves were appressed and this                             was calculated for each treatment and compared
confirmed that the surface of the leaves was                          with the control [17]:
sterile. Single conidia from developing colonies in
the isolation plate were transferred into prepared                        PIMG =                100
standard PDA media to obtain a pure culture.
Agar plugs from single conidia cultures were
used for morphological identification on Malt                         Where, R1= Radial extension of colony in the
Extract Agar (MEA) at x400 magnification of a                         control plate and R2 =Radial extension of colony
compound microscope (OLYMPUS Binocular)                               in sample plate.
                                                       Galley Proof
                                                                      2.5 Effect of Hot              Water      Extract       on
2.3 Effect of Hot Water Extract on Conidia                                Sporulation
    Germination                                                       Agar plugs were taken from three positions on 14
                                                                      days old culture into a McCartney bottle using 1
One mL of different concentrations (15, 30, 45                        cm cork borer and 10 mL of sterile distilled water
and 60% w/v) of the hot water extracts was                            containing 0.05% Tween-80 (surfactant) was
added to 9 mL molten PDA. The plant extract-                          poured into each bottle. The bottle was vortexed
modified PDA was poured into 9 cm Petri dishes                        for 1-2 minutes to dislodge conidia. The
and allowed for 1 hour to solidify. The media for                     concentration of conidia in the suspension was
the control treatment consisted of standard PDA                       estimated using a haemocytometer and the
media alone. The media were inoculated with 10                        density of conidia (conidia cm-2 of the colony)
µL of P. cubensis conidia suspension containing                       was calculated [16].
1.0 x 102 conidia ml-1 prepared from 21 days old
culture and spread-plated using spatula. The                          2.6 Statistical Analysis
Petri dishes were sealed with parafilm to prevent
evaporation of moisture from the agar surface                         Data were subjected to Analysis of Variance
and incubated at ambient temperature for 12                           (ANOVA) where significant difference exists a
hours. Thereafter, sterile coverslips were placed                     Post-Hoc Turkeys Honesty significant difference
in three positions on the surface of the agar and                     was used to separate mean values (IBM SPSS
viewed under x40 objective of compound                                23).
microscope. A conidium with the germ tube
length which was longer than its diameter was                         3. RESULTS
considered as germinated. One hundred conidia
were randomly counted in each of the coverslip                        3.1 Effect of Hot Water Extracts on
field and the percentage germination was                                  Conidia Germination
calculated as:
                                                                      Table 1 shows the effect of different
    % germination =                        100 [16]                   concentrations of the leaf extracts on germination
                                                                      rates of P. cubensis. All the extracts significantly

Falade et al.; ARRB, 31(2): 1-6, 2019; Article no.ARRB.38157

(p≤0.05) inhibited conidia germination when                                significant difference in conidia per colony area
compared with the control. There was 36-9%                                 on all substrates containing the different
inhibition of conidia germination for all the                              concentrations of the extracts. At 15, 30, 45 and
extracts compared to the control that had no                               60% concentrations of S. macrocarpon,
inhibition. Conidia germination with extracts of S.                        sporulation rates were 5.5, 5.4. 5.5 and 5.5 while
macrocarpon at 15, 30, 45 and 60%                                          at the same concentration that of T. diversifolia
concentration was 87, 85, 70 and 62% while that                            the rates were 5.6, 5.6, 5.4 and 5.5 respectively.
of T. diversifolia at same concentrations were 91,
87, 84 and 72%.                                                            4. DISCUSSION             AND       RECOMMENDA-
3.2 Effects of Hot Water Extracts on
    Growth Rate                                                            In this study, all the leaves of the five indigenous
                                                                           plants were air dried and powdered to lower the
Table 2 shows the effect of different                                      surface area thus increasing the rate of reaction.
concentrations of the five leaf extracts on growth                         It has been reported that air dried plant materials
rates of P. cubensis. The growth rate varied                               are less fragile and do not tend to deteriorate an
significantly in relation to plant extracts and their                      advantage which it has over fresh samples [14].
concentration, with values in the control                                  Bioactive constituents are present in varied form
significantly the highest. At 15, 30, 45 and 60%                           in tissues of plant species and can be used as
concentration of extracts S. macrocarpon growth                            natural protectants against diseases [18]. In this
rates were 3.79, 3.65, 3.33 and 2.87 while that of                         study, hot water was used for the extraction
T. diversifolia were 4.25, 4.12, 3.92 and 3.89                             because it is considered as one of the best
respectively. Lower growth rates were recorded                             methods of extraction because it is capable of
at higher concentration of all the extracts used in                        preserving the chemistry of the constituents [19].
the study.
                                                                           In the study, all the extracts of the five plant: T.
3.3 Effects of Hot Water Extracts on                                       diversifolia, U. chamae, C. patens, A. conyzoides
    Sporulation                                                            and S. macrocarpon reduced mycelia growth of
                                                            Galley Proof

                                                                           P. cubensis and the rate of inhibition of growth
Table 3 shows the effect of the five leaf extracts                         was concentration dependent. Highest inhibition
on sporulation of P. cubensis. There was no                                of growth occurred at relatively higher

             Table 1. Effect of hot water extract of five plants on conidia germination
 Concentration      T. diversifolia        U. chamae           C. patens                  A. conyzoides        S. macrocarpon
                       b                     b                   b                          b                     b
 15                 91                     89                  89                         92                   87
                       b                     b                   b                          b                     b
 30                 87                     86                  84                         80                   85
                       b                     b                   c                          c                     c
 45                 84                     81                  73                         73                   70
                       c                     c                   c                          d
 60                 72                     70                  72                         64                   62d
                         a                     a                   a                          a                     a
 Control            100                    100                 100                        100                  100
                   Means with the same letter are not significantly different according to Turkeys test

   Table 2. Effect of four concentrations of hot water extracts of five plants on growth rate of
                                  Pseudoperenospora cubensis
 Concentration       T. diversifolia         U. chame                      C. patens        A. conyzoides        S. macrocarpon
                          b                      b                             b                 b                   b
 15                  4.25                    4.19                          4.07             3.89                 3.79
                          b                      b                             b                 c                   b
 30                  4.12                    4.01                          3.96             3.60                 3.65
                          c                      c                             c                 c                   c
 45                  3.92                    3.88                          3.61             3.30                 3.33
                          c                      c                             d                 d                   d
 60                  3.89                    3.60                          3.30             3.14                 2.87
 Control             4.34a                   4.34a                         4.34a            4.34a                4.34a

                    Table 3. Effect of extract on Sporulation density P. cubensis

 Concentration        T. diversifolia        U. chame                      C. patens      A. conyzoides        S. macrocarpon
                          a                     a                             a               a                    a
 15                   5.6                    5.6                           5.4            5.6                  5.5
                          a                     a                             a               a                    a
 30                   5.6                    5.6                           5.5            5.5                  5.4
                          a                     a                             a               a                    a
 45                   5.4                    5.5                           5.5            5.6                  5.5
                          a                     a                             a               a                    a
 60                   5.4                    5.5                           5.6            5.5                  5.5
                          a                     a                             a               a                    a
 Control              5.9                    5.9                           5.9            5.9                  5.9

Falade et al.; ARRB, 31(2): 1-6, 2019; Article no.ARRB.38157

concentrations of the plant extracts. This was                         anthracnose of papaya, and the study shows that
probably due to increased availability of anti-                        the plant extracts inhibited conidia germination.
fungal chemicals in the medium that was
responsible for suppressing growth. [20]                               The mechanism of some indigenous plants
evaluated the effects of the extracts of                               causing inhibition of mycelia growth and conidia
Mahogany, giant Indian milky weed, garlic and                          germination without significant effect on
ginger at 30-70% concentrations on the growth                          sporulation is not fully understood. There may be
and development of C. gloeosporioides. The                             a need for evaluating composite mixture of plant
study shows that garlic extract at 70%                                 extracts in further studies. Thus, such mixtures
concentration was the most effective. Similarly,                       that have inhibitory effect on growth and
[14] evaluated the antifungal effects of six plant                     germination may produce a more promising
extracts: Blighia sapida, Ricinus communis,                            result on sporulation if applied. The present study
Datura    stramonium,      Tridax   procumbens,                        contribute to the list of researches that extracts of
Jatropha gossypifolia and Sida acuta on the                            the indigenous plant are effective invitro in
mycelia growth of C.lindemuthianum the                                 inhibiting growth of P. cubensis. However, further
pathogen causing anthracnose disease of                                research must be carried out on the field to
cowpea. The result shows that all the plant                            ascertain their effectiveness.
extracts inhibit the growth of the fungus and
efficacy was concentration dependent which                             COMPETING INTERESTS
agree with the current study.
                                                                       Authors have        declared      that   no    competing
In this study, all the five plant extracts at the                      interests exist.
tested concentration did not have any effect on                        REFERENCES
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© 2019 Falade et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License
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provided the original work is properly cited.

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