1. Identification and antifungal activity of pNPP against Peronophythora litchi; the causal agent of downy blight disease of litchi (Litchi chinensis Sonn.)
Uttam Kumar, Srikanti Kumari1, Nirmala Dhar, Arindam Pathak, Aditya kumar, Swastika karki, Rishi raj
Alpine Institute of Management and Technology, Dehradun, 248006 Uttarakhand
1Ranchi University, Ranchi
Introduction
Litchi (Litchi chinensis Sonn.) is one of the most important crop belongs to
the family Sapindaceae (Jiang et al., 2003, Sivakumar et al., 2007, Kumar
et al., 2008). It is indigenous to Southern China (Jiang et al., 2003). Litchi
is an evergreen, tropical and subtropical (Jiang et al.,2003, Sivakumar et
al., 2007, Kumar et al., 2008) fruit tree which bears a whitish translucent
fruit called aril surrounding a single glossy brown seed (Jiang et al., 2003)
and is enclosed inside a pinkish-red rough inedible skin or pericarp which
turns brown and dries out after harvest. Litchi cultivation is specific to
climatic requirement and henceforth, major litchi producing countries in the
world include China, India, Vietnam, Thailand, South Africa, Australia, USA
etc. (Singh and Kaur, 2009). India ranks second in litchi production while
the first place is bagged by China. In India, litchi is cultivated in Bihar,
West Bengal, Assam Jharkhand, Punjab, Chattisgarh, Orrisa, Uttarakhand
and Tripura.
Methodology
Sample Collection and Isolation
Disease affected litchi fruit materials (Fig. 1) were collected from five
different places of Dehradun (Nanda Ki Chawki, Premnagar, Forest
Research Institute, Rajpur Road and Clement Town).
Affected parts from the fruit pericarp were firstly collected and cultured in
Petri plates containing potato dextrose agar (PDA) and on test tubes
containing potato dextrose broth and incubated at 280 C for five days.
Remaining samples were thoroughly washed, dried under the dark
condition and grounded to a fine powder in liquid nitrogen, by using a
mortar and pestle. The powder (plant extract) obtained from each sample
was stored separately and carefully at 20 °C.
The powder obtained from each sample was also separately spread on
petri plates containing PDA and on test tubes containing potato dextrose
broth and incubated at 28 °C for five days. Conidia of the fungus,
Peronophythora litchi was isolated from 4-5 days old culture grown on
PDA media at 28 °C in the dark, was examined under the microscope after
staining with Cotton Blue dye.
Screening of antifungal activity
The control of pNPP on spore germination of Peronophythora litchi was
screened on PDA by petri plate assay. Five different concentrations of
pNPP (0.5. 1.0, 1.5, 2.0 and 2.5 mg/ml) were evaluated for antifungal
activity by Agar Well Diffusion method with a sterile cork borer of size 6.0
mm. The cultures of 48 hours grown on PDA were used for inoculation of
fungal strain on PDA plates. An aliquot (0.02 ml) of inoculums was introduced to
melted PDA and poured into a petri dish by pour plate technique. After
solidification, wells were made on agar plate by using cork borer for diffusion
method. In agar well 0.05 ml of pNPP was introduced. Incubation period of 24 -
48hours at 28°C was maintained for observation of antifungal activity of extract.
The antifungal activity was evaluated by measuring zones of inhibition of fungal
growth surrounding the extracts. The complete antifungal analysis was carried out
under the strict aseptic conditions. The zones of inhibition were measured with
antibiotic zone scale in mm. and the experiment was carried out in triplicates.
Fig. 1 Colonization of isolates LC-1 and LC-2 on Potato
Dextrose agar
Fig.2. Germinating oospore of Peronophythora litchi (A
and B
Fig. 3 Antifungal activity pNPP against
Peronophythora litchi
pNP
P
mg/
ml
Fungal isolates
LC1 LC2 LC3 LC4 LC5
5mg/
ml
10.1 10.4 6.2 6.0 Nil
10m
g/ml
12.0 10.5 5.1 9.0 Nil
15m
g/ml
10.9 12.1 5.3 9.1 1.4
20m
g/ml
15.9 12.2 6.5 10.1 1.1
25m
g/ml
16.4 13.1 10.0 11.0 10.4
Results inhibition was displayed by L
Peronophythora litchi, the causal organism of litchi
downy blight, is a transitional species between
Phytophthora and Peronospora The branched,
determinate sporangiophores of this fungus resemble
those of Peronospora; however, the mode of zoospore
release from sporangia, morphology of sex organs and
ability to grow on artificial media are characteristic of
Phytophphora
Fungus showed colonization on petri plates in the
period of 5 days (Fig. 1). This ability to grow on
artificial media resembles that of Phytophthora.
Germinating oospores of Peronophythora litchi
stained with cotton blue dye examined under the
microscope resembled that of Peronospora or
Phytophthora. The germ tube with many sporangia
(Fig. 2) on the tip emerged from the antheridium,
while that with many sporangia emerged. All
sporangium were connected with the tube. Thus, the
study confirmed the presence of fungus
Peronophythora litchi on litchi fruit causing downy
blight disease of litchi in Dehradun which limits the
production of litchi and its post harvest self-life.
Antifungal activity pNPP against Peronophythora
litchi
Gershon et. al., reported, 18 nitrophenols (2- and 4-
nitrophenols, 4-, 5> and 6-halo-2- nitrophenols, 3-
halo-4-nitrophenols) were tested for antifungal
activity against six fungi (A. niger, A. oryzae, M.
verrucaria, T. viride, M. cirinelloides, and T.
mentagrophytes) in Sabouraud dextrose broth. Only a
small number of halo-nitro and halo-phenols were
tested for antifungal activity. It was recorded that 4-
Fluoro- and 5-fluoro-2- nitrophenols inhibited some
plant pathogenic fungi, among which were Fusarium
sp. and Curvularia sp. (Shirley et. al.,). In the present
study, we have chosen the p-nitrophenol as inhibiting
substrata for In-Vitro control of the Peronophythora
litchii. All the five different concentrations
(0.5mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0mg/ml and
2.5mg/ml) of p-nitrophenol were active against
Peronophythora litchii (Fig. 3). Maximum zone of
inhibition was showed by LC1 (16.4 mm in diameter),
while minimum C 5 (Table 1).