Taxonomic investigation of selected rust fungi using scanning

1. R E S E A R C H A R T I C L E
Taxonomic investigation of selected rust fungi using scanning
electron microscopy from Khyber Pakhtunkhwa, Pakistan
Murtaza Ijaz1
| Rabia Afza1
| Muhammad Zafar2
| Muhammad Hamayun3
|
Shujaul Mullk Khan2
| Zeeshan Ahmad2
| Mushtaq Ahmad2
|
Sumera Afzal Khan4
| Raheem Shah1
| Muhammad Yahya1
1
Department of Botany, Hazara University
Mansehra, Mansehra, Pakistan
2
Department of Plant Sciences, Quaid-i-Azam
University Islamabad, Islamabad, Pakistan
3
Department of Botany, Abdul Wali Khan
University, Mardan, Pakistan
4
Centre of Biotechnology and Microbiology,
University of Peshawar, Peshawar, Pakistan
Correspondence
Rabia Afza, Department of Botany, Hazara
University Mansehra, Mansehra, Pakistan.
Email: ethnopk@gmail.com
Review Editor: Peter Saggau
Abstract
Rusts comprises the largest natural group of plant pathogens including approximately
8% of all described Fungi. Rust fungi are extremely plant pathogens responsible for
great losses to agriculture productivity. Rust species belong to several genera among
which more than half are Puccinia species. In Pakistan, rust causes severe damage to
agriculture crops. Current study was carried out to identify and characterize different
rust species common in the research area through microscopy and Scanning electron
microscopy (SEM) in Khyber Pakhtunkhwa, Pakistan. Morpho-anatomical investiga-
tion of each collected rust species was carried out using different standard protocols.
The dimensions of spores were measured and snapped under a stereomicroscope.
SEM was used to examine the shape, size, and ornamentation of the spores of each
rust fungus. Results revealed documentation of seven rust fungi, that is, Melampsora
euphorbiae, Phragmidium barclayi, Puccinia nepalensis, P. exhausta, P. menthae,
Uromyces capitatus, and Uromyces decorates belong to four different genera, were
recorded. SEM revealed that spermogonia and Aecia were missing in most of the rust
fungus studied. Uredinia was found in a scattered, irregular, lengthy, and epidermis-
enclosed form. Urediniospores were found to be ovulating, elongated, echinulate,
globose to sub-globose, ellipsoid to ovoid, and globose to sub-globose. Telia was
found as sub-epidermal, amphigenous, dispersed, minute, and spherical cells. Telio-
spores ranged in form from cylindrical to oblong. The germ pores were detected in
both apical (top cell) and basal (bottom cell) idiosyncratic and pedicel-attached cells.
The techniques used in the current investigation will aid mycologists in rust identifi-
cation and microscopic characterization.
K E Y W O R D S
Pakistan, Pucciniales, rust fungi, scanning electron microscopy, taxonomy
1 | INTRODUCTION
Rust fungi (Basidiomycota/Pucciniales) are very diverse class of obli-
gate pathogen due to their wide range of natural spore dispersal
behavior (Hibbett et al., 2011). Rust fungi damage and reduce the
accumulated biomass, particularly in major economic crops
(Mueller, 2011). Rust fungi are placed in order Uredinales of Basidio-
mycetes and epitomized for bearing basidia and basidiospores
(Cummins & Hiratsuka, 2003). The rust fungi received their name due
to rust-colored mobs of urediniospores (or Uridinia) produced on the
host's plant. Approximately only 8,400 (8%) rust species
(Pucciniomycotina) have been described till now from estimated
Received: 7 January 2021 Revised: 13 September 2021 Accepted: 14 September 2021
DOI: 10.1002/jemt.23947
Microsc Res Tech. 2022;85:755–766. wileyonlinelibrary.com/journal/jemt © 2021 Wiley Periodicals LLC. 755

2. 98,998 recorded species of fungi (Kirk et al., 2001). These are catego-
rized into 163 genera of 14 families (Kirk et al., 2001; Ono &
Aime, 2006). Rust fungi are grouped within the subphylum
Pucciniomycotina that are parasites of not only the advanced mono-
cots and dicots but ferns as well (Aime et al., 2014).
The relationship of rust fungi with their host is very specific
(Kendrick, 2000). Some of the rust species needed two taxonomically
diverse hosts for the completion of their life cycle. This type of life
cycle is called macrocyclic/heteroecious, while some require only one
specific host. They attack specific genera or plant family. Hence, caus-
ing severe damage to crop or plant species may lead to economic loss.
For example, Puccinia graminis cause wheat rust, Cronartium ribicola
causes white pine blister rust and Hemileia vastatrix causes coffee
leaf rust.
Rust species have a complex lifecycle comprised of 5–6 various
spore types. Teliospores are present where karyogamy takes place
that rises basidia by germination. Meiosis takes place in basidia that
gave rise to four basidiospores. However, the fruition of the sexual
cycle happens in different rust species that may lead to the formation
of aeciospores and urediniospores (Cummins & Hiratsuka, 2003).
Rust fungi have been found to attacked typically in a pattern of
irregular patches of infections or tumors (Hernández & Hennen,
2003). Approximately 8.4 million/ha (70%) of wheat cultivated areas
in Pakistan are vulnerable to rust attack. Due to such a remarkable
influence on cash crop production, rust fungi acquired specific atten-
tion globally (Afzal et al., 2008). The correlation of rust fungi to a dis-
tinctive host plant is very significant in ecology and plant pathology,
causing financial loss, especially to agriculture and horticulture (Aime
et al., 2006).
More recently, scanning electron microscopy (SEM) has made a
massive impact in diverse branches of biological sciences particularly,
the identification of macro and microflora (Ali et al., 2021; Ayaz
et al., 2020; Caglayan, 2020; Gul et al., 2020; Hadidchi et al., 2020;
Sabbah et al., 2020). However, SEM is rarely used in literature for the
identification and characterization of Rust fungi.
Rust fungi of Khyber Pakhtunkhwa are the least explored and so
far only 174 rust species are explored (Ishaq, 2017). Recently, two
species of rust fungi were reported from Shangla and Abbottabad,
District of Khyber Pakhtunkhwa, Pakistan (Ishaq et al., 2011; Ishaq
et al., 2020). Beside few reports on rust fungi from Khyber
Pakhtunkhwa, rust attacks are very common in this region. Current
study was an attempt to explore rust fungi in the area for updating
record and future control measures. For this purpose, rust infected
plants were collected from different parts of Khyber Pakhtunkhwa
and the specimens were studied under stereomicroscopy and scan-
ning electron microscope to identify the new races of rust fungi.
2 | MATERIALS AND METHODS
The rust infected plant specimens were collected from different locali-
ties of Khyber Pakhtunkhwa, Pakistan (Figure 1; Tables 1 and 2) and
poisoned with Mercuric Chloride (HgCl2) solution. Spores were
collected from specimens that were at different stages of infection.
Spore maturity was determined based upon the areas of sporangia
becomes swollen and are seen as small. When mature they spores are
thus seen externally, break through the walls and at this time spore
collection was made.
The herbarium hands sections materials and spores were
mounted on a slide glass slide with lactophenol, followed by gentle
heating.
2.1 | Microscopic characterization
The spore organizations were observed and photographed under a
Stereomicroscope on 25–50 amplification (Han et al., 2020; Khan
et al., 2020). A maximum of 30 spores thoroughly examined for all
spore stages, that is, from smallest to the largest sores in each speci-
men (Aime et al., 2017). The apparent spores feature, that is, shape,
size, and ornamentation were further studied under the Scanning
electron microscope (Model: JSM5910-JEOL, JAPAN). Measurements
of teliospores include apical thickness. In uredinia and aeciospores,
spore shape and size, characteristics of reticulum surface, location and
number of germ pores, spore apex shape and thickness, and morphol-
ogy of the spore base and spore wall were analyzed. The reticulate
FIGURE 1 Location map of sampling sites for the exploration of
Rust Fungi in Khyber Pakhtunkhwa
756 IJAZ ET AL.

3. ornamentation includes spore size and wall thickness. For each speci-
men, the spore occurring spore states were noted (Table 2). The digi-
tal images were created, filtered, and saved on a computer
(de Oliveira Souza et al., 2020; Gonzalez Woods, 2000).
Consequently, all the samples were illustrated and elucidated sci-
entifically. The depictions were match up with the available manuals
for accurate characterization of rust fungi (e.g., Ahmad, 1956; Ahmad
et al., 1997; Iqbal et al., 2008; Cummins Hiratsuka, 2003). To con-
firm their scientific entity where some generic/species names have
been updated with currently accepted name, literature for example,
Aime et al. (2017), Aime and McTaggart (2020), Cummins and
Hiratsuka, 2003, and websites on rust fungi Species for example,
Fungorum (www.speciesfungorum.org) and MycoBank (www.
mycobank.org) were consulted.
TABLE 1 Sampling Sites description of the study area in KP, Pakistan
Site description Nathiagali Mansehra Shangla Shabaz Ghari Kaghan Dir upper
Champion
et al., 1965.
Himalayan
subtropical,
moist
temperate, and
subalpine
forests
Humid
subtropical
pine forest
Subtropical
pine forests
and the
subalpine
forests
Sami arid
tropical–
subtropical
forests
Himalayan
moist
temperate
and
subalpine
forests
Sub-tropical to
alpine regions
having dry and
coniferous dry
temperate
forests
Climatic
conditions
Rainfall (mm) 1,616 1,532 1,400 559 1,489 1,500
Humidity (%) 68.5 57 66 45 67 60
Temperature
C
(annual mean)
12 15.3 C 15 32.5 13.9 17
Coordinates 34.0729
N
73.3812
E
34.3313
N
73.1980
E
34.8872
N
72.7570
E
34.2355
N
72.1594
E
34.7794
N
73.5270
E
35.3356
N
72.0468
E
Altitude (m) 2,375 1,082 1,300–3,000 1,000–2,056 2,026 m 2,000–3,000
Edaphic factors Clay—loamy Shallow,
loamy
Loamy—Clay
loam
Clay, slightly
loam
Clay loam Shallow slightly
gravely loams/
silt loams
TABLE 2 Rust species collection data from host plants in the study area
S. no.
Voucher
no. Host plant Family Rust fungi Locality GPS data Collected by
1 6332 Euphorbia helioscopia L. Euphorbiaceae Melampsora euphorbiae
(Ficinus C. Schubert)
Castagne, 1843.
Nathiagali 34
040
2500
N
73
220
50.2700
E
8,077 ft
Rabia Afza
Murtaza
Ijaz
2 6333 Rubus niveus Thunb. Rosaceae Phragmidium barclayi
Dietel, 1890
Shabaz
Ghari
34
140
08.6200
73
090
35.0700
1,041 ft
Murtaza Ijaz
3 6335 Rumex nepalensis
Spreng
Polygonaceae Puccinia nepalensis Barclay
Dietel, in Dietel, Hedwigia
29, p. 265 (1890)
Kaghan 34
460
41.7200
N
73
310
35.9900
E
6,925 ft
Rabia
Afza
4 6337 Clematis grata Wall. Ranunculaceae Puccinia exhausta
Dietel, 1900.
Shangla 34
520
5900
N
72
430
36.7800
E
6,789 ft
Raheem
Shah
5 6338 Mentha longifolia (L.) L. Lamiaceae Puccinia menthae Pers., Syn.
meth. fung. (Göttingen)
1:227 (1801)
Mansehra 34
20 10.0900
N
73
12 42.1500
E
3,853 ft
Murtaza Ijaz
6 6341 Chenopodium album L. Amaranthaceae Puccinia menthae Pers. Shahbaz
Garhi
34
140
4100
N
72
090
30.3600
E
1,033 ft
Murtaza Ijaz
7 6345 Desmodium elegans DC. Leguminosae Uromyces capitatus Syd. P.
Syd., Öst. bot. Z. 52(5):
182 (1902)
Dir upper 35
210
24.8900
N
71
590
10.6800
E
7,875 ft
Muhammad
Yahya
8 6346 Lespedeza juncea subsp.
sericea (Thunb.)
Steen
Fabaceae Uromyces decorates
Syd. P. Syd., Annls mycol.
5(6): 491 (1907)
Mansehra 34
20 10.0900
N
73
12 42.1500
E
7,451 ft
Murtaza Ijaz
IJAZ ET AL. 757

4. TABLE 3 Quantitative data recorded from stereo-microscopic examination of rust fungi
Species name Uredinia Urediniospores Telia Teliospores
Euphorbia helioscopia L.
(Euphorbiaceae)
Rust
Genera = Melampsora
Scattered, amphigenous,
yellowish
Globose to sub globose,
ellipsoid, echinulate,
yellowish.
Size 17–20 12–17.5 μm
Sub epidermal,
amphigenous,
brownish black or
black
Cylindrical oblong, apex not
thick, yellowish brown size
35–65 10–15 μm
Rubus niveus Thunb
(Rosaceae)
Rust
Genera = Phragmidium
Light brown
Abaxial, scattered size 0.5–
0.7 μm diameter
Ellipsoid or ovoid and
yellowish.
Wall thickness is 1–1.5 μm
Size 10–13.5 13.5–
20 μm
Dark brown
Hypophyllous
Scattered or in group
Size 0.4–2 mm in
diameter
Shape = oblong, cylindrical,
sometime attenuated at the
base, 4–7 septate, very
rarely 3 septate, apex 4–
6 μm thick, size 22–
32.5 72.5–115 μm. Wall
thickness 2.5–3.5 μm
yellowish brown pedicel
hyaline 17.5–20 μm broad
to 137 μm long
Rumex nepalensis
Spreng. (Polygonaceae)
Rust
Genera = Puccinia
Scattered, amphigenous,
naked spores, and
yellowish to pale brown
Globose, ovoid, or rounded,
echinulate, size 21–
24 μm 18–22 μm.
Diameter 1–2 μm
Brown color
Shape-ellipsoid,
rounded both ends
Rounded ellipsoid at the apex
to the septum, constricted
at septum.
Reddish brown in color. Size is
23–35 μm 15–20 μm
Wall thickness is 1–1.4 μm.
Pedicel 3–4 μm
Clematis grata wall
(Ranunculaceae)
Rust Genera = Puccinia
Shape is small rounded
scattered or in groups.
Dark brown to blackish
Rounded small oval shape
or ovoid shape
Light brown.
Size 17–21 μm 12–
16 μm
Small, scattered, or in
circular groups
Amphigenous
Dark brown to blackish
Mostly ellipsoid, sometimes
single celled elongated,
(conical or round at the
apex) base mostly rounded.
Apex wall thickness 2–3 μm
sometimes not thickened,
slightly constricted at the
septum and are not smooth.
Pedicel 82 μm in length.
Hyaline are deciduous. Size
15.7–24.6 μm 26.5–
50.2 μm in length
Mentha longifolia (L.)
L. Rust
Genera = Puccinia
Amphigenous scattered or
in groups
Pale reddish brown in color.
0.5–0.4 mm in width
Globose to sub globose
Ovoid, echinulate
Yellowish to pale brown.
Size 14–25 μm 21–
27.5 μm
Minute to large.
Scattered or in groups.
Dark brown to reddish.
Size 1–0.9 mm width
Broadly ellipsoid, slightly thick,
not constricted at the
septum. Pale brown to dark
brown in color.
Pedicel 7 μm long
Apex is near to the upper cell
while in case of lower cell
near to the septa. Size 22.5–
42.5 μm 17.5–24.5 μm
Desmodium elegans DC.
(Leguminosae)
Rust Genera = Uromyces
Abaxial, scattered form,
yellow to brown.
Size 0.2–0.4 μm
Globose or ellipsoid,
minutely echinulate.
Light yellow.
Size = 17–
20.2 μm 20.3–23.1 μm
Wall thickness = 1–1.5 μm
Abaxial, scattered,
sometimes in group.
Color dark brown.
Size = 0.2–0.3 μm in
diameter
Globose. Reddish brown in
color. Hyaline short broken
down.
Size = 17.1–25.3 μm 22.4–
33.5 μm
Wall thickness = 1.5 μm.
Pedicel = 45 μm
Lespedeza juncea
Subsp. sericea (Thunb.)
Steen
Rust Genera = Uromyces
Amphigenous
Spread or in compact form,
minute. Light to dark
brown. Size = 1–13 μm
Globose to sub globose or
oblong, echinulate.
Color is light brown.
Size = 13.3–
16.3 μm 19.1–21.1 μm
Wall thickness = 1–1.2 μm
Globose or sub
globose
Cylindrical, brown to
chestnut brown
Height 4 μm, globose or
cylindrical, apex with papilla
(hyaline) chestnut brown.
Wall thickness 13.2–
20.2 16–26.1 μm wall.
Hyaline 1.4 μm. Pedicels are
deciduous
758 IJAZ ET AL.

5. 3 | RESULTS
A total of seven rust fungi that is, Melampsora euphorbiae,
Phragmidium barclayi, Puccinia nepalensis, P. exhausta, P. menthae,
Uromyces capitatus, and U. decorates belong to four different gen-
era were identified and characterized using microscopic tech-
niques (Table 3). The detailed description of each species was as
follows:
3.1 | M. euphorbiae (Ficinus C. Schubert)
Castagne, 1843
This rust species was reported on Euphorbia helioscopia
L. (Euphorbiaceae) at II–III stages from Pakistan at an elevation of
8,077 ft from Nathiagali (Ayubia National Park). The Microscopic
and SEM studies revealed that the spermogonia and aecia were
absent in the M. euphorbiae. Uredinia were irregular and dispersed
with yellowish-brown. The Urediniospores echinulate, globose/
sub-globose, golden brown and 10–23 23–25.5 μm in size.
Paraphyses thick-walled, capitates, and 15–24.5 μm at the apex
(Figure 2). Telia subepidermal, amphigenous, and brownish black.
Teliospores were yellowish to brown and cylindrical to oblong in
shape.
3.2 | P. barclayi Dietel, 1890
This rust species was reported on Rubus niveus Thunb (Rosaceae) at stages
II and III at an elevation of 1,041 ft from Shahbaz ghari. The Aecia and
Spermagonia were absent in this species. Uredinia were present in form
of patches, irregular, abaxial, and 0.5–0.7 mm in diameter. Urediniospores
ellipsoid to ovoid (9.2–13 12.5–19 μm) with 1–1.5 μm wall thickness.
Telia was recorded as dark brown, hypophyllous, and 0.3–2.1 mm in diam-
eter. Teliospore septate (4–6), cylindrical or oblong (2–31.5 72–
115 μm) with 4–6 μm apex thickness, thickened wall (2–3 μm), hyaline
pedicel (17–20 μm width and 137 μm length (Figure 3).
3.3 | P. nepalensis Barclay and Dietel, in Dietel,
Hedwigia 29: 265 (1890)
This rust species was reported on Rumex nepalensis Spreng.
(Polygonaceae). This species' SEM showed that the uredinia
FIGURE 2 Morphology of Melampsora euphorbiae on Euphorbia helioscopia L. (a) Diseased host plant, (b,c) distribution pattern of teliospores
and uredinospores, (d) Urediniospores and Teliospores comprehended with echinulate surface ornamentation, (e) Urediniospores showing
echinulate surface ornamentation
IJAZ ET AL. 759

6. were scattered, irregular, long, and enclosed by the epidermis.
Uredinospores were globose, ovulate, or elongated and
echinulate type with 21–25 μm 18.5-23 μm possessing three
germ pores. The teliospores ellipsoid, circular at the end, hyaline
(papilla), next to the septum (24–35.5 30–44 μm), thickened
wall (1.1–1.4 mm). Pedicel hyaline with 3–7.5 μm in length
(Figure 4 and Table 3).
3.4 | P. exhausta Dietel, 1900
This fungus was recorded on Clematis grata Wall (Ranunculaceae)
at 6,789 ft altitude from Shangla. The SEM of P. exhausta com-
prehended uredinia was small spherical, either irregular (scattered)
or in round clusters enclosed by epidermis with 0.5–0.8 μm length.
The length of urediniospores varies from 17–21 12–16 μm.
Telia minute, amphigenous, irregular, or in clusters form covered
by epiderms. Teleospores prolonged, cylindrical, round at apex,
tapering, thick apical wall (2–3 μm), smooth, hyaline to yellowish
and light brown, and 15.7–24.6 26.5–50.2 μm long. The germ
pores were present on both apical and basal sides with 82 μm in
length (Figure 5).
3.5 | P. menthae Pers., Syn. meth. fung. (Göttingen)
1: 227 (1801)
This fungus was reported on Mentha longifolia (L.) L. from Mansehra at
3,853 ft elevation. P. menthae has Uredinia on the abaxial side, how-
ever, in some cases amphigenous with echinulate (1.5–2.1 μm) wall,
irregular (scattered) or in the form of clusters (16–20.2 μm 21–
28 μm) and pale brown. Urediniospores were ovoid, globose, ellipsoid
(19–23.5 24–29 μm in length). Telia was amphigenous, scattered in
cluster form, elongate, smallest to largest, and brown to black. The
teliospores were ellipsoid (20–24.5 24–29.5 μm), apex size up to
10 μm, prominent germ pores, reddish to a dark brown wall, minute
varicose, hyaline (8 85 μm) (Figure 6).
3.6 | U. capitatus Syd. P. Syd., Öst. bot. Z. 52(5):
182 (1902)
This rust species was recoded on Desmodium elegans DC. (Leguminosae)
from Dir upper at 7,875 ft elevation. Both the spermogonia and
aecia were absent in this rust species. The Uredinia were abaxial,
irregular with 0.2–0.4 μm in diameter. Urediniospores were
FIGURE 3 Phragmidium barclayi pakistanica on Rubus niveus. (a) Infected leaves of hostplant by Phragmidium barclayi. (b) Teliospore.
(c) Urediniospores. (d–f) Scanning microphotographs of Uredinia and Telia on abaxial leaf surface of host plant. Teliospores with verrucose
surface ornamentation
760 IJAZ ET AL.

7. recorded as an ellipsoid, globose, meticulously echinulate (17–
20.2 21–23.1 μm), indistinct germ pore, and the thickened wall
(1–1.6 μm). Telia recorded on abaxial side, scattered (some time
present in groups form having diameter 0.2–0.3 μm diameter).
Teliospores globose, subglobose (16.1–25.3 μm 22.4–33.5 μm),
thickened wall (1.7 μm), short pedicel and hyaline up to 44 μm
(Figure 7).
3.7 | U. decorates Syd. P. Syd., Annls mycol. 5(6):
491 (1907)
This species of rust fungi was reported on Lespedeza juncea subsp.
sericea (Thunb.) Steen (Fabaceae) from Mansehra at 7,451 ft elevation
(Table 2). The U. decorates has uredinia amphigenous, opaque brown,
and irregular in shape. Uredinia are small with 1–1.3 μm in length.
Urediniospores subglobose (13.3–16.3 μm 19.1–21.2 μm), wall 1–
1.2 μm in length, and germ pores (3–4) hyaline in color. Teliospores
4 μm in length, cylindrical, globose, apex hyaline (papilla). Pedicels
were deciduous/hyaline with 1.2–4.2 7.1–17.5 μm in length
(Figure 8 and Table 3).
4 | DISCUSSION
Rust is the distinctive unit of fungi with a particular group of individ-
uals. It is comprised of obligate parasites with a complex lifecycle that
is, five spore stages and a very narrow range of hosts. Approximately
8,400 (8%) rust species have been identified till now (Aime
et al., 2014). The Microscopic investigation is generally important in
the identification of fungi because it easily distinguishes the internal
structure and spore shape and arrangements of each fungal species.
The current study revealed the identification and characterization of
rust fungi using a scanning electron microscope. It showed that
spermogonia and Aecia were mostly absent in the majority of the
recorded rust species.
The current research project revealed the identification of seven
rust species belongs to five different genera including Puccinia (three
Species), Uromyces (two species), Melampsora (one species), and
Phragmidium (one species).
Puccinia is the leading and biggest genus of Pucciniales (3,000–
4,000 species) parasitizing vascular plants particularly Polygonaceae,
Asteraceae, Poaceaea, Liliaceae, and Cyperaceous in all regions except
polar. This genus is characterized by typically two-celled teliospores,
FIGURE 4 Puccinia oenanphes on Rumex nepalensis. (a) Diseased Leaf of host-plant. (b) Stereomicroscopy of Telia sori. (c) Teliospore. (d–f)
Scanning electron microphotographs of teliospores and urediniospores showing echinulate and verrucose surface ornamentation
IJAZ ET AL. 761

8. with exceptional cases of one or three-celled in some species
(Cummins Hiratsuka, 2003). From Pakistan till date 27 taxa belong-
ing to genus Puccinia were recorded on 29 different host plants from
different localities.
We reported first time P. menthae and P. exhausta on M. longifolia
and C. grata, respectively from Shangla Hills and Mansehra Pakistan.
Both taxa have previously been reported on M. longifolia (L.) L., Origa-
num vulgare subsp. vulgare L., Calamintha umbrosa (M. Bieb.) Fisch.
Meyer, Ind., C. vulgare L., M. longifolia, Nepeta campestris Benth.,
Potentilla and Rumex species (i.e., Afshan et al., 2011; Ahmad, 1956;
Ahmad et al., 1997; Ishaq, 2017) from different localities of Pakistan.
Our result revealed the lack of spermogonia and aecia in
P. menthae and P. exhausta recorded on M. longifolia and C. grata,
respectively is similar to that observed by Edwards et al. (1999) and
Ali et al. (2016) by using microscopic, molecular and SEM techniques
in their examination. Like our study, it also comprehended Uredinia on
the abaxial side, Uredeniospores ovoid, globose and ellipsoid in shape
Telia was amphigenous, scattered in cluster form, elongate, smallest
to largest, and pulverulent brown to black.
Melampsoraceae, a monophyletic family of heteroecious rusts
producing aecia, generally on telia on members of Euphorbiaceae and
Salicaceae mainly, and on needles of conifers (Cummins
Hiratsuka, 2003). Till date from Pakistan 10 species of this genus are
described that is, M. euphorbiae (Ficinus C. Schub.) Castagne,
M. ciliata Barclay, M. populnea (Pers.) P. Karst., M. capraearum Thum.,
M. epitea Thüm., M. lini (Ehrenb.) Thüm., M. laricis-populina Kleb.,
M. populina subsp. populina (Jacq.) Lév., M. yoshinagae Henn, and
Magdalis salicis-albae Kleb (Ishaq, 2017). However, all these records
are based on morphological characters (Ahmad et al., 1997;
Ishaq, 2017).
Generally, host identification and morphological characteristics
are considered as key oddities to identify the Pucciniales, however,
this approach is not ideal for Melampsora spp. as all species of this
genus are distinctive morphology (Ishaq, 2017). Additionally, species
identifications on the basis of host plant data cannot be appraised
precisely for majority of the taxa of this genus, as a single Melampsora
species can infect several species of host plants in one genus or a sin-
gle host plant can be infected by different species of Melampsora
(Aime et al., 2017).
Our study recorded M. euphorbiae rust on E. helioscopia from
Nathiagali (Ayubia National Park). This was the first attempt of recording
rust species from Ayubia National Park, Pakistan. We reported the
FIGURE 5 Puccinia exhausta on Clematis grata. (a) Infacted host plant. (b) Infected leaf of host plant showing telium with teliospores. (c,d)
Teliospores and uredioniospores. (e) Scanning microphotographs of Uredinia and Telia on abaxial leaf surface of host plant. (f,g) Teliospores.
Comprehended uredinia spherical shape either irregular or in round clusters
762 IJAZ ET AL.

9. FIGURE 6 Puccinia menthae on Mentha longifolia. (a). Diseased leaves. (b,c) Stereomicroscopy of telia sori. (d) Scanning microphotographs of
teliospores and urediniospores ornamentation. (e) Teliospore. (f) Urediniospores with echinulate surface orientation
FIGURE 7 Morphology of Uromyces capitatus. (a) Diseased leaves of Desmodium elegans by fungus Uromyces capitatus. (b) Telial sori under
stereomicroscope. (c) Urediniospores. (d) Teliospores. (e,f) Scanning microphotographs of Teliospores with reticulate surface ornamentation
IJAZ ET AL. 763

10. morphology of Urediniospores as echinulate, that are analogous to the
results of other researchers, who examine the same species
(i.e., Cummins Hiratsuka, 2003; Damadi, 2014; Maier et al., 2003).
Other characteristics of this species included capitate paraphyses, naked
Uredinia, crust-like telia with single-celled Teliospores. Our results are in
close harmony with Zhao, Wang, Tian, and Kakishima (2015), who also
examined the genus Melampsora characters via SEM.
From Pakistan, taxon M. euphorbiae has previously been recorded
and identified based on disease pattern and spore morphology, on
E. hypericifolia L. from Gilgit, Karachi, and Muzaffarabad (Ahmad
et al., 1997), on leaves of E. helioscopia L., Euphorbia dracunculoides Lam.
and E. cornigera Boiss from other regions of Pakistan (i.e., Ahmad, 1956;
Afshan et al., 2011; Fiaz, Khalid, Ahmad, 2013 Ishaq, 2017). How-
ever, it is a new record for Ayubia National Park, Pakistan.
Phragmidium genus is characterized by the presence of one to
several celled teliospores having smooth and more often verrucose
surface ornamentation is type genus of Phragmidiaceae. The genus
mainly inhabits the northern hemisphere and mostly infecting species
of Rosaceae, predominately of genus Rubus L., Rosa L., and Potentilla
L. (Cummins Hiratsuka, 2003). From Pakistan previously 19 species
of Phragmidium have been reported. Species was typified by the
presence of three germ pores per cell and hygroscopic pedicels and
2–9 celled teliospores with long pedicle.
During a field survey to the forests of Shahbaz ghari, P. barclayi
was on R. niveus from for first time, added to new records from the
studied area. Furthermore, Paspalum barclayi has been recorded on
R. niveus as well in the moist subtropical region of Pakistan (Fiaz
et al., 2013). We examined the ph. Barclayi on under electron
microscopy along with stereo microscopy. Other species of
Phragmidium have been also reported on Triticum aestivum L.,
Hordeum vulgare L., and Dactylis glomerata L. (Ishaq, 2017). Liu et al.
(2020) also reported the three different species of Phragmidium on
different species of Rosaceae that is, Rosa laevigata Michx. Rosa
abietina Gren. ex H.Christ, Rosa banksiae R.Br., Rosa bracteata J.C.
Wendl., R. fujisanensis (Makino) Makino, Rosa luciae Franch.
Rochebr., and Rosa willmottiae Hemsl based on morphological
description and description.
Uromyces is a major genus of Pucciniales after Puccinia Pers, para-
sitizes different plants especially economically important plants world
widely. In this genus special character is present which makes them dif-
ferent from other these characters are the presence of aecia, subepi-
dermal, spermogonia, singly borne urediniospores on pedicles that
FIGURE 8 Morphology of infected host plant by Uromyces decorates. (a) Infected leaves of Lespedeza juncea (Fabaceae). (b) Telial sori under
stereomicroscope. (c) Teliospore. (d) Urediniospores. (d–f) Scanning microphotographs of teliospores and urediniospores scattered (irregular) and
amphigenous ornamentation
764 IJAZ ET AL.

11. possess assorted germ pores, one-celled pigmented, teliospores, which
possess solo germ spores. This genus consists of almost 600 described
species throughout the world (Cummins Hiratsuka, 2003). We exam-
ined and documented two species of Uromyces genus that is,
U. capitatus on D. elegans from Dir upper (temperate region) and
U. decorates on L. juncea from Mansehra (sub-tropical region).
U. capitatus Syd. P. Syd. on D. elegans DC. Has verrucose,
rounded teliospores and pale papillae. This is the first ever report of
U. capitatus from Dir Upper, Pakistan, an addition to fungal flora of Dir,
Pakistan. Previously from Pakistan, U. capitatus has been reported on
leaves of Desmodium tiliaefolium G. Don. from Salt Range, Sharan, and
Swat (Ahmad, 1956) and on D. elegans from Leepa Valley, Azad Jammu,
and Kashmir (Khalid Iqbal, 1996, Saba et al., 2011). Similarly, seven
species of genus Uromyces have been identified and examined for mor-
phological and molecular characteristics by Ishaq (2017) from northern
areas and different localities of KP, Pakistan.
One of the important applications of the current research investi-
gation is the use of SEM to identify and characterize rust fungi.
Uredinia were present in dispersed, irregular, long, and enclosed by
the epidermis. Urediniospores were recorded as ovulate, elongated,
echinulate, globose to subglobose, and ellipsoid to ovoid. Telia was
present as sub-epidermal, amphigenous, scattered rarely, minute, and
spherical. Teliospores were present from cylindrical to oblong in
shape. The germ pores were recorded both in apical (upper cell) and
basal (lower cell) form distinct and contiguous to the pedicel. Similar
techniques were also used by other researchers to identify and char-
acterize various groups of fungi (Aime et al., 2006; Ali et al., 2016; Ali
et al., 2017; Liu et al., 2020).
5 | CONCLUSION
A total of seven rust fungi species were identified using scanning elec-
tron microscopy. The rust species M. euphorbiae, P. barclayi,
P. nepalensis, P. exhausta, P. menthae, U. capitatus, and U. decorates
were found on E. helioscopia, R. niveus, Rumex, C. grata, M. longifolia,
D. elegans and L. juncea, respectively. Uredinospores and Teliospores
were common in all the isolated rust fungi. This report shows that the
forest of selected sampling sites has immense potential of fungal
diversity. A thorough exploration of each site is further needed to
explore more diversity. The pathogens identified in this study can be
used as a biological control agent against weeds in economically
important plantation. This is first attempt to use EMS for taxonomic
study of rust in Pakistan. The data generated in this study can be used
for comparisons in future on large scale to determine the exact status
of these fungal pathogens of economically and ecologically important
plants of Pakistan. For example, fungus M. euphorbiae recorded in the
present study has been already screened for use as a biological con-
trol against the weeds and has clear potential as a biological control
agent particularly against Euphorbia esula L. and E. cyparissias L. in the
USA (Bruckart Dowler, 1986) other Euphorbia species.
Use of essential microscopic techniques (LM and SEM) has devel-
oped a wide variation in spore morpho-types observed through
microscopic examination could contribute to accurately identify the
species. Therefore, techniques that is, SEM used along with
steromicroscopy in the current study will further help in the identifica-
tion of rust fungi and other fungi in general. The use of SEM method-
ology in the current study will help examine the correlation of fungi
on the surface of different vascular flora, increasing the possibilities of
advanced examination of rust taxonomy on different host, and to exe-
cute precise transmission of comprehensive and wide-ranging taxo-
nomic classification of rust fungi.
ACKNOWLEDGMENT
The authors are highly thankful to the Central Resources Laboratory,
Department of Physics, University of Peshawar, Pakistan, for provid-
ing the facility of Scanning Electron Microscopy.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were cre-
ated or analyzed in this study.
ORCID
Rabia Afza https://orcid.org/0000-0001-9710-7039
Muhammad Zafar https://orcid.org/0000-0003-2002-3907
Shujaul Mullk Khan https://orcid.org/0000-0002-6531-572X
Mushtaq Ahmad https://orcid.org/0000-0003-2971-2848
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