Indian Phytopath.

70 (4) : 446-451 (2017)

DOI 10.24838/ip.2017.v70.i4.76988

RESEARCH ARTICLE

Morphological and molecular variability of Podosphaera

pannosa causing rose powdery mildew in Himachal Pradesh,
India

VIJAY KUMAR* and SUNITA CHANDEL

Department of Plant Pathology, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan 173 230, Himachal Pradesh,
India

Received: 24 October 2017/ Accepted: 5 December 2017/ Published online: 22 January 2018
© Indian Phytopathological Society 2017

ABSRACT: Rose powdery mildew caused by Podosphaera pannosa (Wallr.) de Bary is one of the most serious and devastating
diseases of roses grown worldwide including Himachal Pradesh in natural as well as protected cultivation. Different isolates
of six districts were morphologically distinguished from one another with respect of size of mycelium, conidia and
conidiophores which ranged between 4.0 to 4.8, 22.8 × 12.4 and 72.0 to 78.0 µm, respectively. Molecular characterization of
ten isolated from six districts were studied with seven RAPD markers. The dendrogram revealed similarity index among ten
fungal isolates ranged between 19 to 73 per cent with an average of 46 per cent. Maximum similarity of 73 per cent was
observed between isolate-5 and isolates-8 of district Mandi Himachal Pradesh. Least similar was isolate-7 which had 19 per
cent similarity with other isolates.

Keywords: Dendrogram, isolates, RAPD marker, similarity index

Rose is the one of the most important ornamental flower
which has the high economical value as a cut flower. It is
also important as a loose flower for its uses in various
ornamentations, food values and in various medicinal
purposes. Roses are infected by various plant pathogenic
microorganisms like viruses, bacteria and fungi. Most
important fungal foliar diseases are downy mildew, black
spot, grey mould, leaf spot and rust (Linde and Shishkoff,
2003) which causes heavy losses to roses. Among these
fungal diseases powdery mildew is most destructive and
devastating. The powdery mildew fungus is observed on
the upper surface of the leaves, but it also infect the lower
surface of leaves, young shoots, stems, buds and flowers
(Gastelum et al., 2014).
In India the occurrence of rose powdery mildew for
the first time was reported from Kashmir, Ranikhet and
Dehradun (Butler and Bisby, 1931) and from Rajasthan
by Pathak (1967). The occurrence of rose powdery
mildew caused by the Podosphaera pannosa has been
reported worldwide from USA (Longree, 1939; Yarwood,
1944; Mence and Hildebrandt, 1966), United Kingdom
(Radclyffe, 1861; Howden, 1968; Price, 1970), China (Leu
and Kao, 1975), Netherland (Hajlaoui and Bélanger,
1991), British Columbia (Ng et al., 1997) and Italy (Pasini
et al., 1997) and many other countries. Woronichine had
made a division in 1914 in which Sphaerotheca pannosa
var. rosae infecting roses and S. pannosa var. persicae
infecting peach and almond were two varieties of
*Corresponding author: vnarwal777@yahoo.com
pathogens recognized (Horst, 1983). Rose powdery
mildew is caused by Podosphaera pannosa (Wallr.: Fr.)
de Bary (syn. Sphaerotheca pannosa (Wallr.: Ex Fr.) Lev.)
is the major fungal pathogen of roses grown in
greenhouses and also one of the most important
diseases of outdoor roses worldwide (Hajlaoui et al.,
1991; Linde and Shishkof, 2003; Agrios, 2005; Leus et
al., 2006; Horst and Cloyd, 2007). Anamorphic stages of
Sphaerotheca and Podosphaera were not distinguishable
among each other when the surface of conidia was
observed with a scanning electron microscope (Cook et
al., 1997) and the difference between Podosphaera and
Sphaerotheca is only made by the host plant. The results
of ITS data was complied by Takamatsu et al. (1998)
and the combination of morphological data with lTS
sequencing (Saenz and Taylor, 1999) supported the
theory that both genera are congeneric. It was concluded
that all Sphaerotheca species belong to the Podosphaera
genus.
The present study was carried to ascertain variability
among the various isolates collected from the different
regions of Himachal Pradesh.
MATERIALS AND METHODS
Morphological variability
Variation in the different isolates of fungi was studied on
the basis of shape and size of the mycelium, conidia
and conidiophores.
Indian Phytopathology 70 (4) : 446-451 (2017) 447

DNA extraction and RAPD analysis
For molecular characterization, DNA was isolated using
the CTAB method (Murray and Thompson 1980) with
some modifications. Purified DNA was run on a 0.7%
agarose gel with diluted uncut lambda DNA (25 ng/µl)
as standard to assay its concentration and integrity by
ethidium bromide fluorescence. The quantification of
DNA was done with help of UV/VIS spectrophotometer
(BIORAD Smart spec 3000, Gurgaon, Haryana). The
quantified DNA samples were diluted in TE buffer to make
a final concentration of 50 ng/µl for PCR reactions. RAPD
reactions were performed according to the protocol of
Williams et al. (1990) with the modifications to enhance
reproducibility and consistency of RAPD profiles. RAPD
amplifications were performed in 25 µl of reaction volume
containing 1x PCR buffer (containing 15 mM MgCl2), 2.5
mM each dNTP (Sigma, USA), 100 pmol of 20 random
decamer primers (Operon Technologies Inc, USA), 1 unit
of Taq DNA polymerase (Banglore Genei, Banglore,
India) and 50 ng genomic DNA template. PCR
amplifications were performed in a thermal cycler (Perkin
Elmer Thermal Cycler GeneAmp® PCR System 9700
Model, Grand Island, NY 14072, USA) with heated lid
technology for 45 cycles. The PCR conditions were: initial
denaturation of genomic DNA at 95°C for 4 min followed
by 45 cycles of DNA template denaturation at 94°C for 1
min, primer annealing at 32°C for 1 min, DNA
amplification at 72°C for 2 min and final primer extension
at 72°C for 8 min. Amplicons were separated on a 1.5%
agarose gel prestained with ethidium bromide solution
using 1 X TAE buffer. The gels were run for 2 h at 60 V
and the RAPD amplicon profiles were recorded using
Syngene Gel Documentation System with GeneSnap
software. The size of the amplified fragments was
determined using1000 bp plus ladder (MBI Fermentas,
Lithuania) and Gene- Tools software. All RAPD reactions
were performed twice to test the reproducibility of the
amplicon profiles. Twelve numbers of primers (designed
by Sigma-Aldrich) i.e. OPE-07 (AGATGCAGCC), OPV-
02 (AGTCACTCCC), OPD-15 (CATCCGTGCT), OPM-
18 (CACCATCCGT), OPC-12 (TGTCATCCCC), OPE-03
(CCAGATGCAC), OPF-01 (ACGGATCCTG), OPH-02
(TCGGACGTGA), OPH-07 (CTGCATCGTG), OPF-03
(CCTGATCACC), OPG-03 (GAGCCCTCCA) and OPP-
01 (CAGGCCCTTC) were used for characterization.
RESULTS AND DISCUSSION
Variability studies
Morphological characterization: Different isolates
collected from the different rose growing areas of
Himachal Pradesh during survey period of two years
2015 and 2016, were characterized morphologically. It
is evident from the Table 1 that district wise collected
isolates (B, K, M, S, SR, SO) were morphologically
different among each other on the basis of shape and
size of conidia, conidiophores and mycelium. The
average size of mycelium, conidia and conidiophores
varied from 4.0 to 4.8 µm, 22.0 × 11.8 to 22.8 × 12.4 µm
and 72.0 to 78.0 µm, respectively. Superficial, hyaline,
septate, branched mycelium was observed in different
fungal isolates. The highest width of mycelium was
observed in the fungal isolates of district Solan with 4.8
µm followed by 4.7 µm and 4.6 µm of district Sirmour
and Mandi, respectively. The lowest width of mycelium
was noticed in fungal isolates of district Kangra 4.0 µm
followed by Bilaspur and Shimla with mycelial width of
4.3 and 4.4 µm, respectively. Cylindrical to ovoid,
aseptate, hyaline conidia were found in different fungal
isolates with variation in the size. The smallest size

Table 1. Morphological characterization of different isolates of Podosphaera pannosa

District Isolates Fungal Structure Shape Size (µm)

Bilaspur B Mycelium Superficial, hyaline, septate, branched 3.2 to 6.0 (4.3)

Conidia Cylindrical to ovoid, aseptate, hyaline 20.2 to 26.0 x 11.3 to 13.4 (22.2 x 12.2)
Conidiophore Upright stalk, hyaline, aseptate, unbranched 52.0 to 94.3 (74.2)
Kangra K Mycelium Superficial, hyaline, septate, branched 3.0 to 6.2 (4.0)
Conidia Cylindrical to ovoid, aseptate, hyaline 20.2 to 26.4 x 11.4 to 13.0 (22.0 x 11.8)
Conidiophore Upright stalk, hyaline, aseptate, unbranched 52.26 to 94.6 (72.0)
Mandi M Mycelium Superficial, hyaline, septate, branched 3.6 to 6.8 (4.6)
Conidia Cylindrical to ovoid, aseptate, hyaline 20.8 to 26.4 x 11.6 to 13.7 (22.5 x 12.3)
Conidiophore Upright stalk, hyaline, aseptate, unbranched 54.4 to 96.6 (74.4)
Shimla S Mycelium Superficial, hyaline, septate, branched 3.4 to 6.4 (4.4)
Conidia Cylindrical to ovoid, aseptate, hyaline 20.6 to 26.2 x 11.5 to 13.8 (22.4 x 12.4)
Conidiophore Upright stalk, hyaline, aseptate, unbranched 54.2 to 96.2 (74.0)
Sirmour SR Mycelium Superficial, hyaline, septate, branched 3.8 to 6.6 (4.7)
Conidia Cylindrical to ovoid, aseptate, hyaline 20.6 to 26.4 x 11.3 to 13.8 (22.6 x 12.4)
Conidiophore Upright stalk, hyaline, aseptate, unbranched 54.8 to 96.3 (76.0)
Solan SO Mycelium Superficial, hyaline, septate, branched 3.6 to 6.9 (4.8)
Conidia Cylindrical to ovoid, aseptate, hyaline 20.4 to 26.6 x 11.8 to 13.6 (22.8 x 12.4)
Conidiophore Upright stalk, hyaline, aseptate, unbranched 53.2 to 98.3 (78.0)
448
conidia of 22.0 × 11.8 µm, 22.2 × 12.2 µm and 22.4 ×
12.4 µm were recorded in district Kangra, Bilaspur and
Shimla, respectively. Whereas the biggest size of conidia
(22.8 × 12.4 µm, 22.6 x 12.4 µm and 22.5 × 12.3 µm)
were observed in isolates collected from district Solan,
Sirmour and Mandi (Fig. 1).
Hyaline, upright stalks, aseptate and unbranched
types of conidiophores were observed in different fungal
isolates with variation in size. Longest conidiophores
were reported from district Solan (78.0 µm) followed by
76.0 µm and 74.4 µm from Sirmour and Mandi,
respectively. Smallest conidiophores were reported from
district Kangra 72.0 µm followed by Bilaspur (74.2 µm)
and Shimla (74.0 µm). These results are in conformity
Fig. 1. Morphological variation in conidia of rose powdery mildew of different districts of Himachal Pradesh (A) Bilaspur; (B) Kangra; (C)
Mandi; (D) Shimla; (E) Sirmour; (F) Solan
Indian Phytopathology 70 (4) : 446-451 (2017)
with Gastelum et al. (2014) who reported 4.7 to 6.0 µm
mean diameter of mycelium, cylindrical to ovoid conidia
with 25.8 to 30.4 ìm long and 13.9 to 17.3 ìm wide. Similar
findings were registered by Faheem et al. (2016) who
reported mycelium as hyaline, septate, branched,
measuring 3.4-6.8 µm (average 5.1 µm) in width,
conidiophores with upright stalk’s, hyaline, aseptate and
conidia of egg shaped, aseptate, hyaline, measuring 20.7
to 26.9 × 11.8-13.0 µm (average 23.8 × 12.4 µm) in size.
Molecular characterization: Genetic diversity was
studied among the ten isolates [2 isolates (no. 4, 10)
from Bilaspur, 1 isolate (no. 3) from Kangra, 2 isolates
from Mandi (no. 5, 8), 1 isolate from Shimla (no. 9), 2
isolates (no. 1, 2) from Solan, 1 isolate (no. 6) from
Indian Phytopathology 70 (4) : 446-451 (2017)
Fig. 2a. RAPD profile of isolates 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10
with primers OPE-07 and OPM-18
Fig. 2b. RAPD profile of isolates 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10
with primer OPD-15 and OPH-02
Sirmour and 1 wild rose isolate (no. 7)] collected from
different rose growing regions of Himachal Pradesh using
RAPD-PCR technology. Twelve random oligonucleotide
primers (Operon technologies) were used and only seven
primers were able to amplify the genomic DNA
successfully. A total of fifty eight RAPD bands were
produced with seven primers, out of which fifty six were
polymor phic and only two were monomor phic.
Percentage of total polymorphism obtained with primers
was 96.55 per cent and average number of bands per
primer was 8.29 as shown in Table 3. Total number of
RAPD band varied from 5-12 with minimum of 5 bands
in OPM-18 and maximum of 12 bands in OPH-02
followed by the OPE-03 (10). Total number of polymorphic
bands varied from 5 to 12 with minimum of 5 bands in
OPM-18 and OPF-03 and maximum of 12 bands in OPH-
02 followed by the OPE-03 (10).
449
Fig. 2c. RAPD profile of isolates 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10
with primer OPF-03 and OPC-12
Fig. 2d. RAPD profile of isolates 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10
with primer OPE-03
Genetic relatedness among all the isolates of
Podosphaera pannosa
The similarity coefficient revealed that the similarity
among all the isolates ranged between 19 to 73 per cent,
with an average of 46 per cent (Fig. 3). The dendrogram
(Fig. 3) of ten isolates of Podosphaera pannosa divided
into two different clusters. First cluster again divided into
two sub clusters. First sub-cluster consisted of isolates
from district Solan i.e. isolate-1 (Nauni) and isolate-2
(Ramshahar), Sirmour i.e. isolate-6 (Chakhal), Bilaspur
i.e. isolate-4 (Kotipura) and isolate-10 (Chharol), Mandi
i.e. isolate 5 (Rajgarh) and isolate-8 (Behal) and Shimla
i.e. isolate-9 (IIWBR-Flowerdale). The second sub-cluster
consists of only one isolate-3 (Bhattu) from district
Kangra. Similarly, the second cluster consisted of one
isolate i.e. isolate-7 which collected from the wild grown
roses of Deodhar area of district Mandi. However, among
all the combinations, maximum similarity was found
between the isolate-5 and isolate-8 of district Mandi
(73%), while the isolate-5, isolate-8 and isolate-9
collected from Rajgarh, Behal, IIWBR-Flowerdale were
450 Indian Phytopathology 70 (4) : 446-451 (2017)

Fig. 3. Genetic relatedness among ten isolates viz. 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 of Podosphaera pannosa based on combined analysis
of seven random primers; *Bilaspur (isolates 4 and 10), Kangra (isolate 3, Mandi (isolates 5 and 8), Shimla (isolate 9), Solan
(isolates 1 and 2), Sirmour (isolate 6) and wild rose (isolate-7)

Table 2. Total number of amplified fragments and polymorphic fragments generated by using 7 decamer primers

Primers Fungal isolates Total number of Total number of monomorphic Total number of polymorphic
Amplification bands amplified bands amplified bands amplified

OPE-07 I 1-10 9 1 8
OPM-18 I 1-10 5 0 5
OPD-15 I 1-10 except I 7 9 0 9
OPC-12 I 1-10 except I 7 7 0 7
OPE-03 I 1-10 except I 3,4,8 10 0 10
OPH-02 I 1-10 except I 5, 8 12 0 12
OPF-03 I 1-10 6 1 5

Table 3. Summary of RAPD amplified product obtained from
test isolates
Total number of primer studied
Total number of polymorphic primer
Total number of RAPD bands
Total number of polymorphic bands
Total number of monomorphic bands
61 per cent similar to each other. However, minimum
similarity was found in isolate-7 (19%) with other isolates.
Molecular evaluation of the ten isolates of the P.
pannosa revealed that the isolate-1, isolate-2 and isolate-
6 fall in the same cluster which might be due to the reason
that both isolate-1 and isolate-2 were collected from the
same region i.e. Solan having the same environmental
growing conditions and isolate-6 slightly different from
the later two might be due to slight shift in the
geographical region and morphological studies also
revealed that shape and size of conidia, mycelium and
conidiophores of these isolates were very similar to each
other. Isolate-5 and isolate-8 has highest similarity with
12
each other because they were collected from district
Mandi and their collection regions were located very near
7
to each other and the pathogen is exposed to same
58 environmental conditions. Isolate-9 from Shimla region
56 which is much colder than other regions and maybe there
2 is difference in the virulence of pathogens and
morphological data also supports these results because
fungus from these regions showed the similar characters
with each other i.e. shape and size of mycelium, conidia
and conidiophores. Isolate-3 was very different from the
other isolates of cluster-1 and morphologically the fungus
from Kangra district has smallest size of mycelium,
conidia and conidiophores. Isolate-7 was very different
from all other isolates which may be due that this isolate
was collected from wild grown rose revealing difference
in virulence of pathogen and difference in host
germplasm.
Several similar studies has been carried in powdery
mildew for assessing variability using RAPD markers like
Indian Phytopathology 70 (4) : 446-451 (2017)
six sets of specific primers were detected by using 10
RAPD primers (McDermott et al., 1994). In a similar way
five RAPD and two SCAR markers were used for
detecting variability in Erysiphe graminis f. sp. hordi by
Caffier (1999). On the other hand, Sheikholeslam et al.
(2005) successfully used RAPD-PCR experiment to
investigate genetic diversity of 30 isolates of populations
of Erysiphe betae , the causal agent of sugar beet
powdery mildew in Iran. Al-Sadi et al. (2012) studied the
variability among the 35 isolates of P. pannosa using four
pairs of AFLP primers and found to have low levels of
gene diversity, which suggests that P. pannosa has been
recently introduced into Oman. Genetic similarity of 11
to 26 per cent was reported by Devi and Prakasam (2015)
between the different isolates of Leveillula taurica
infecting chilli by using RAPD markers.
It has been concluded from the present study that
all the isolate collected from different districts of Himachal
Pradesh were morphological and as well as molecularly
different from each other may be because of different
growing environmental condition, difference in
germplasm which is studied and difference in virulence.
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