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ORIGINAL ARTICLE
Morpho-molecular variability and host reactivity of Albugo candida
isolates infecting Brassica juncea genotypes in India
Oinam Washington Singh
1
&Naveen Singh
2
&Deeba Kamil
1
&Vaibhav Kumar Singh
1
&Thokala Prameela Devi
1
&
Lakshman Prasad
1
Received: 14 January 2020 /Accepted: 15 October 2020
#Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2020
Abstract
To generate information on variability in the pathogen causing white rust disease in Brassica species and their ability to infect
Brassica juncea genotypes, thirteen isolates of Albugo candida infecting B. juncea and one isolate of Wilsonia bliti infecting
Amaranthus blitum were collected from different rapeseed-mustard growing regions of India. These isolates were characterized
based on different morphological data (sporangial shape and size; shape, size, and color of the pustule) and molecular data
(cytochrome c oxidase subunit II (COX2) of mtDNA and internal transcribed spacer (ITS) region of rDNA). All the sporangia
were globose or spherical to ellipsoidal with a size ranging from 199.01 μm
2
to 357.23 μm
2
. White to creamy colored pustules
were observed with a diameter ranging from 0.5–3.0 mm. Sequence analysis revealed that the Indian isolates of A. candida
collected from B. juncea showed close identity with the Korean isolates infecting B. juncea. W. blilti isolate collected from
A. blitum form a distinct clade with A. amaranthi infecting Amaranthus spinosus from Korea. Further, the pathogenic reaction of
ten A. candida isolates was recorded on thirty genotypes of B. juncea that exhibited differential responses to the tested isolates.
The three genotypes, PDZ-3, Pusa Karishma, and Donskaja were found to be resistant against all the ten isolates, which can
further be used as a resistance source. PDZ-2 genotype was found to be susceptible only against the Ac-Hr isolate while the
genotype BioYSR shows a susceptible reaction against Ac-Skn and Ac-Wltn isolates only. This set of thirty genotypes was able
to differentiate the ten isolates of A. candida into four physiological races (AcRBj1 to AcRBj4) based on the disease reaction. The
information generated through this study will be helpful for further study on identification and molecular mapping of resistance
gene(s), their deployment over time and space, and, thus, better management of white rust disease in B. juncea.
Keywords Genetic diversity .Races .Host differentials
Introduction
Oilseed crops are the second most important determinant of
the agricultural economy, next to cereals. Of these, Brassica
species comprising B. rapa, B. juncea, B. napus, and
B. carinata are one of the most important rabi oilseed crops.
The production of rapeseed-mustard has been increasing rap-
idly across the globe due to its ever-escalating demand for
edible oils and its by-products (Kumar and Chauhan 2005).
Rapeseed-mustard plants are cultivated in 53 countries spread-
ing over 6 continents across the globe, covering an area of
30.06 million hectares (mha) with a total production of
55.97 million tonnes (mt) and a productivity of 1.8 t per hect-
are (t/ha) (Anonymous 2017). India contributes about 18.26%
area and 11.68% production of rapeseed-mustard in the world,
which makes it the third-largest producer of rapeseed-mustard
after Canada and China. These four species (B. juncea, B.
rapa, B. carinata and B. napus) arecultivatedinanareaof
5.76 mha with a production of 6.82 mt during 2015–16 with
the major area occupied in the states of Rajasthan, Madhya
Pradesh, Uttar Pradesh, Haryana, Assam, Jharkhand and
Gujarat (Anonymous 2017). Among rapeseed-mustard, the
Indian mustard (B. juncea) occupies about 80% of the total
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s42161-020-
00690-4.
*Lakshman Prasad
laxmanprasad25@yahoo.com
1
Division of Plant Pathology, ICAR-Indian Agricultural Research
Institute, New Delhi 110012, India
2
Division of Genetics, ICAR-Indian Agricultural Research Institute,
New Delhi 110012, India
Journal of Plant Pathology
https://doi.org/10.1007/s42161-020-00690-4
area in India and it is the second-largest oilseed crop next to
soybean in terms of area and production (Vinu et al. 2013).
The estimated requirement of total vegetable oil by 2030 is
about 29.05 mt of which 14.29 mt is expected to be contrib-
uted by rapeseed-mustard only (Vision 2050 2015). The low
productivity of rapeseed-mustard is attributed to its suscepti-
bility to different biotic and abiotic factors. Among the
rapeseed-mustard group of crops, Indian mustard possesses a
better level of tolerance against most of the biotic and abiotic
stresses (Gupta 2011), however, diverse mustard growing
conditions in different geographical areas still demand a better
level of tolerance. White rust or the white blister rust disease
caused by A. candida is one of the most important biotic
constraints in the production of B. juncea worldwide
(Saharan and Verma 1992). The pathogen infects both vege-
tative and reproductive parts of the plants. Infection on inflo-
rescence leads to the formation of staghead galls that lead to
flower sterility and no seedformation which accounts for most
of the yield losses (Verma and Petrie 1980). In India, white
rust disease causes 17–34% yield reduction of B. juncea due
to staghead formation but sometimes caused up to 60–80%
yield loss when infection occurred in both leaf and inflores-
cence simultaneously (Saharan et al. 1984;LakraandSaharan
1989). Among the different management strategies, the devel-
opment of resistant cultivars is one of the most effective, eco-
nomic, and eco-friendly approaches. Unfortunately, all the
available varieties of mustard are susceptible to one or other
races of A. candida (Kolte 1985). White rust resistance has
been reported in Indian mustard genotypes, however, cultivars
with a desirable level of resistance, which is effective across
locations, could not be developed and deployed due to lack of
information on the presence and distribution of A. candida
races, and their ability to infect B. juncea genotypes.
A high degree of diversity and complexity was report-
ed within the A. candida species due to its wide host
ranges with an ability to infect up to 63 genera and 241
different plant species (Biga 1955; Gupta and Saharan
2002;Choietal.2007; Mishra et al. 2009). This pathogen
is highly specialized, and several biological races
(biotypes) have been identified and classified based on
specificity to different cruciferous species. Previous stud-
ies have suggested that the Brassica-Albugo specificity
occurs at the levels of the genus, species within a genus,
or even among cultivars within a species (Pidskalny and
Rimmer 1985;Hilletal.1988;Petrie1988;Rimmeretal.
2000; Saharan 2010). Six races of A. candida were iden-
tified by Pound and Williams (1963) based on the host
species from where the pathogen has been primarily iso-
lated. Later, race 7 and race 8 were added by Verma et al.
(1975) and Delwiche and Williams (1977) which are iso-
lated from B. rapa and B. nigra respectively. Petrie
(1994) identified two new races isolated from B. napus
and B. rapa cv. Reward from Canada and Kaur et al.
(2008) identified two races infecting B. juncea and
Raphanus raphanistrum from Western Australia. In
India, 49 different races have been identified based on
their differential reactions against various Brassica hosts
including the wild relatives (Singh and Bhardwaj 1984;
Lakra and Saharan 1988;BhardwajandSud1988;Verma
et al. 1999;Jat1999; Gupta and Saharan 2002). But, the
major limitation in the proper racial discrimination is the
unavailability of an internationally standardized set of dif-
ferential hosts of each cruciferous species.
With the advancement in molecular biology, sequence
analyses of the COX2 region of mtDNA and ITS region of
rDNA were found to be highly conserved and useful in
resolving closely related species or isolates within the spe-
cies in the phylum Oomycota (Cooke et al. 2000;Göker
et al. 2004; Thines et al. 2009;Hudspethetal.2003;
Petkowski et al. 2010). Genetic diversity of the pathogen
was investigated by the sequence analysis of COX2 and
ITS region from A. candida isolates collected from differ-
ent hosts by Choi et al. (2006). Based on the sequence data,
a high degree of genetic diversity was confirmed and the
possibility of a species complex within the A. candida was
addressed. A new species, Albugo laibachii was reported
based on a concatenated phylogenetic tree from the ITS and
COX2 genes which specifically infects only Arabidopsis
thaliana (Thines et al. 2009).ITSsequence analysis of nine
Australian A. candida isolates showed a close association
with other previous isolates from Australia, Europe, and
Asia except for the isolate infecting B. tournefortii which
forms a separate clade on its own (Kaur et al. 2011).
Petkowskietal.(2010) studied 31 Australian A. candida
isolates collected from 11 hosts using the rDNA ITS re-
gion, rDNA LSU (large subunit) region, and mtDNA
COX2 region. It was observed that the majority of
Australian isolates were the common form of A. candida
except for Cardamine hirsuta, which belongs to a previ-
ously reported but undescribed species. Although different
nuclear and cytoplasmic regions of the pathogen were re-
ported to use for better understanding the genetic diversity
in different countries, the detailed molecular studies on
Indian isolates of A. candida under a single host species
has not been studied so far.
So, the present study was conducted to understand the
complexity within A. candida isolates infecting B. juncea col-
lected from different geographical locations of India.
Morphological traits such as sporangial shape and size, pus-
tule shape, size and color, and two molecular markers, COX2
of mtDNA and ITS of the rDNA region were used to analyze
the morphological and genetic diversity of A. candida species
under a single host B. juncea. Further, thirty genotypes of
B. juncea were tested against ten A. candida isolates to check
the nature of virulence and their differential response against
the tested genotypes.
J Plant Pathol
Material and methods
Collection and preservation of white rust diseased
samples
Thirteen isolates of A. candida infecting B. juncea and one
isolate of W. blilti infecting A. blitum were collected during
winter (rabi) season of 2014, 2015 & 2016 from different
geographical locations representing most of the mustard cul-
tivating areas of India (Table 1). Collected leaf samples were
stored at -40 °C before use.
Purification and maintenance of white rust inoculums
The seeds of susceptible cultivar Varuna (B. juncea) were
used to raise seedlings for multiplication of A. candida in
plastic pots (15 cm dia.) containing sterilized soil. After ger-
mination, thinning was done and ten plants per pot were main-
tained in the glasshouse. Pathogen inoculums of different iso-
lates were prepared by scraping a single pustule of the respec-
tive reference isolate with a sterile scalpel into a sterilized
1.5 ml eppendorf tube. Then, 1 ml of sterile distilled water
was added to the tube to make sporangial suspension and kept
at 4 °C for 4 h for zoospore release. The concentration of
zoospores was adjusted to 8 × 10
4
zoospores/ml using a he-
mocytometer (Sachan et al. 2004). The seedlings were drop
inoculated with 10 μl zoosporangial suspension onto the ad-
axial surface of every lobe of each cotyledon at 8 days after
sowing (DAS) using a micropipette. To promote infection, the
inoculated plants were kept under the enhanced humidity
(>98%) for 72 h in a high humidity chamber sealed with
plastic sheets and free water (up to 2 cm height) maintained
at the bottom of the chamber. After three days, the plants were
taken out from the chamber and kept outside in controlled
conditions at 18 °C and 13 °C day and night temperatures,
respectively. The diseased plant leaves with the pustules were
collected at 14 days post-inoculation (dpi) and stored at -40 °C
for further studies.
Morphological variability
The sporangial shape was recorded on 25 sporangia per pus-
tule in all the isolates under the 40X magnification in the
digital light microscope. Sporangial sizes were recorded (in
μm) from ten sporangia per pustule for different isolates at
40X magnification in the digital light microscope. The size
and shape of pustules were recorded by taking visual obser-
vationsof25pustulesfromeachA. candida isolates. The size
of the pustules was recorded (in mm) by measuring the diam-
eter of 10 random pustules from each sample with the help of a
scale. The color of pustules for each isolate was also observed
visually from the infected samples.
Test for significance for qualitative traits
To validate the morphological data, a matrix was prepared by
using binary value (0–1) for three qualitative traits i.e. pustule
shape, pustule color, and sporangial shape, where a value of 1
indicates the presence of the particular trait and a value of 0
represents the absence of that trait. Jaccard’s similarity coeffi-
cients were employed to construct the phenetic tree using the
‘NTSYS’software (Rohlf 1992).
Test for significance for quantitative traits
For quantitative data i.e. pustule diameter and sporangial
sizes, Duncan’s multiple range tests were used for checking
the significant difference between the average diameter of
pustules and the average area of sporangia among the different
isolates of A. candida collected from different locations of the
country. For this, the sporangial area was calculated by using
the mathematical formula for an ellipse.
Area of sporangia ¼πx Major radius RðÞx minor radius rðÞ
Duncan multiple range tests;Rp ¼Qp S2p=n
1=2
Principle of component analysis (PCA)
In order to find out the relative importance of the morpholog-
ical data in creating the variation in Indian A. candida isolates,
PCA was performed based on the three qualitative and two
quantitative characters by using the SYSTAT-13 statistical
software (SYSTAT Inc. 2009).
Genetic variability analysis
DNA extraction
Genomic DNA was extracted from all the isolates of Albugo
species under study using host leaf tissue with pustules fol-
lowing the cetyl trimethyl ammonium bromide (CTAB) meth-
od (Lee and Taylor 1990). Infected leaf samples (0.5 g) were
grounded in a sterilized mortar and pestle using liquid nitro-
gen (-196 °C) and transferred to 1.5 ml eppendorf tubes. Six
hundred microliter of preheated 2X CTAB extraction buffer
[2% (w/v) CTAB +100 mM Tris-HCl + 1.4 M NaCl +20 mM
EDTA with a pH of 8.0] was added and incubated at 60 °C for
one hour in water-bath. An equal volume of
chloroform:isoamyl alcohol (24:1) was added and centrifuged
at 10,000 rpm for 20 min at 24 °C. The aqueous phase was
separated and transferred into a fresh tube. A 0.6 volume of
ice-cold isopropanol and 0.1 volume of sodium acetate buffer
(3 M) were added and incubated at -20 °C for overnight. The
next day, DNA was precipitated by centrifuging at 10,000 rpm
JPlantPathol
for 10 min at 4 °C. The pellet DNA was collected and washed
twice with 75% ethanol, dried, and resuspended in 70 μlTE
buffer.
Polymerase chain reaction and sequencing
To study the molecular diversity, the mitochondrial COX2
gene was amplified by using the forward (5’-GGC-AAA-
TGG-GTT-TTC-AAG-ATC-C-3′) and reverse (5’-CCA-
TGA-TTA-ATA-CCA-CAA-ATT-TCA-CTA-G-3′) primer
(Hudspeth et al. 2000). Nuclear rDNA region containing the
partial 18S gene, both internal transcribed spacers (ITS1and
ITS2) and the 5.8S gene was amplified by using the primer
DC6 (5′-GAG-GGA-CTT-TTGGGT-AAT-CA-3′)(Cook
et al. 2000) and LR0 (5’-GCT-TAA-GTT-CAGCGG-GT-3′)
(Moncalvo et al. 1995). PCR reactions were conducted in
Table 1 Indian isolates of A. candida collected from B. juncea hosts along with the retrieved sequences from NCBI used for comparison
Sl. No. Isolate
code*
Host species Pathogen species Place of collection Gene bank accession
number
References
COX2 ITS
1–Arabis alpina Albugo candida Romania GU292086 –Ploch et al. 2011
2 Ac-Bgpt Brassica juncea Albugo candida Uttar Pradesh, India MG193580 MT649867 This study
3Ac--MghyBrassica juncea Albugo candida Meghalaya, India MG193581 MT649868 This study
4Ac-CbrBrassica juncea Albugo candida West Bengal, India MG193582 MT649866 This study
5Ac-BhrtBrassica juncea Albugo candida Rajasthan, India MG193583 MT649869 This study
6Ac-KulBrassica juncea Albugo candida Himachal Pradesh, India MG193584 –This study
7Ac-GwlrBrassica juncea Albugo candida Madhya Pradesh, India MG193585 –This study
8Ac-Hr Brassica juncea Albugo candida Haryana, India MG193586 MT649863 This study
9Ac-Jpr Brassica juncea Albugo candida Rajasthan, India MG193587 MT649872 This study
10 Ac-Kt Brassica juncea Albugo candida Rajasthan, India MG193588 –This study
11 Ac-Pnt Brassica juncea Albugo candida Uttrakhand, India MG193589 MT649864 This study
12 Ac-Skn Brassica juncea Albugo candida Uttar Pradesh, India MG193590 MT649865 This study
13 Ac-Wltn Brassica juncea Albugo candida Tamilnadu, India MG193591 MT649871 This study
14 Ac-Dli Brassica juncea Albugo candida Delhi, India MT672346 MT649870 This study
15 –Brassica juncea Albugo candida Korea, Namyangju AY927046 AY929826 Choi et al. 2006
16 –Brassica juncea Albugo candida Korea, Jeju AY927047 AY929828 Choi et al. 2006
17 –Brassica juncea Albugo candida Korea, Namyangju AY913810 AY929827 Choi et al. 2006
18 –Brassica oleracea var.
gemmifera
Albugo candida Forth, Australia GQ328860 GQ328823 Petkowski et al. 2010
19 –Biscutella laevigata Albugo candida Valais, Switzerland DQ418506 DQ418494 Thines et al. 2009
20 –Berteroa incana Albugo candida Krems, Austria DQ418508 DQ418495 Thines et al. 2009
21 –Capsella bursa-pastoris Albugo candida Zuid, Netherlands DQ643944 DQ643916 Thines et al. 2009
22 –Capsella bursa-pastoris Albugo candida Washington, USA DQ643952 DQ643919 Choi et al. 2007
23 –Diplotaxis erucoids Albugo candida Kiriat-Anabim, Palestine –DQ418496 Choi et al. 2006
24 –Diplotaxis tenuifolia Albugo candida Spain DQ418518 DQ418497 Choi et al. 2006
25 Diplotaxis tenuifolia Albugo candida Malda GU292029 –
26 Eruca sativa Albugo candida Daudkhel, Pakistan DQ418514 DQ418503 Choi et al. 2006
27 Eruca sativa Albugo candida Australia GU292095 –
28 –Heliophila meyerii Albugo candida Vanrhynsdorp, South
Africa
DQ418515 DQ418493 Thines et al. 2009
29 –Iberis amara Albugo candida California, USA DQ418522 DQ418499 Thines et al. 2009
30 –Lunaria sp. Albugo candida USA AY913797 AY929840 Choi et al. 2006
31 –Raphanus sativus Albugo candida Seoul, Korea AY927059 AY929841 Choi et al. 2006
32 –Raphanus sativus Albugo candida Kangnung, Korea AY927060 AY929842 Choi et al. 2006
33 –Raphanus sativus Albugo candida Busan, Korea AY913801 AY929843 Choi et al. 2006
34 –Sisymbrium leteum Albugo candida Pyongchang, Korea AY927061 AY929845 Choi et al. 2006
35 –Sisymbrium leteum Albugo candida Pyongchang, Korea AY913808 AY929844 Choi et al. 2006
36 –Sisymbrium loeselii Albugo candida Bulgaria AY913802 AY929846 Choi et al. 2006
37 –Sisymbrium sp. Albugo candida Australia –GQ328822 Petkowski et al. 2010
38 –Thalpsi arvense Albugo candida NY, USA AY913809 AY929847 Choi et al. 2006
39 –Amaranthus spinosus Wilsonia amaranthi Chunchon, Korea AY913805 AY929824 Choi et al. 2006
40 Wb-Bbnr Amaranthus blitum Wilsonia bliti Odisha, India MT672347 –This study
*Ac- Albugo candida,Wb-Wilsonia bliti, Bhrt-Bharatpur, Dli-Delhi, Bgpt-Baghpat, Mghy-Meghalaya, Pnt-Pantnagar, Wltn-Wellington, Hr-Hisar,
Cbr-Cooch Behar, Jpr-Jaipur, Kt-Kota, Kul-Kullu, Gwlr-Gwalior, Skn-SK Nagar
J Plant Pathol
50 μl reaction volumes, with each reaction tube containing
2μl of template DNA (200 ng/μl), 5 μl of 10X buffer
[50 mM KCl, 100 mM Tris-HCl (pH 8.0), 0.1% Triton
X-100, 20 mM MgCl
2
], 1.5 μlof10mMdNTP,1μleach
of forward and reverse primer(100 pM primers), 0.5 μlofTaq
polymerase (5 unit/μl), and 39 μl of double-distilled water.
The PCR mix was kept for initial denaturation at 95 °C for 5
mins followed by 35 cycles of denaturation for 1 min at 95 °C,
annealing for 1 min at 58 °C, and extension for 2 min at 72 °C
and a final extension for 10 min at 72 °C. The PCR amplified
products were resolved by electrophoresis on 1.2% agarose
gel prepared in 1X TAE buffer (pH 8.0) at 85 V for 45 mins.
All the samples with the distinct bands were selected for se-
quencing through (outsourcing) Sci-Genome Labs Pvt. Ltd.,
Kerala, India.
Sequence alignment and phylogenetic analysis
Fourteen COX2 and ten ITS sequences with a nucleotide
length of 531 and 829 bases, respectively, were used for se-
quence alignments along with the other A. candida sequences
retrieved from the NCBI for comparison (Table 1). Clustal W
algorithm was used to perform the multiple sequence align-
ment. The phylogenetic tree was constructed in MEGA 7.0
software using maximum likelihood (ML) analysis with a
bootstrapvalue of 1000 (Kumar et al. 2016). Albugo
amaranthi from A. spinosus was used as an outgroup while
constructing the tree.
Host reaction test
A total of thirty B. juncea genotypes were used to study
the differential pathogenic reactions against ten isolates
of A. candida under controlled conditions (Table 3).
Isolates from Kullu, Himachal Pradesh, and Cooch
Behar, West Bengal were not included in the study since
the B. juncea is not commercially cultivated in these
areas. Instead, B. napus and B. oleracea is cultivated in
Himachal Pradesh, whereas, only B. rapa is predomi-
nantly cultivated in the plains of West Bengal.
Inoculum suspension was prepared by following the
same procedure as mentioned above. Both lobes of each
cotyledon were drop inoculated (10 μl per drop) with the
zoospore suspension using a micropipette at 8 DAS. Ten
isolates were inoculated on a set of thirty genotypes of
B. juncea as mentioned above along including a suscep-
tible cultivar Kranti. The disease reaction on each geno-
type was recorded at 10 dpi using a rating scale of 0–6
developed by Conn et al. (1990) and diseased plants
were collected at 20 dpi along with the pustules and
stored at -40 °C for further use.
Rating scale (0–6) for the disease index (Conn et al.
1990)
Rating scale Leaf area (%) covered
by the pustules
Disease reaction
0 No symptoms Immune response
11–5 Highly resistant
25–10 Resistant
310–20 Moderately resistant
420–35 Moderately susceptible
535–50 Susceptible
6 More than 50 Highly susceptible
Documentation of races among A. candida obtained
from B. juncea
A system on wheat rusts in India and the system proposed in
other countries are already available for racial documentation
(Nagarajan et al. 1983). We have proposed a system of thirty
host differentials including a global white rust-resistant culti-
var, Donskaja along with other B. juncea genotypes for vari-
able reaction to white rust disease. Our set also contained
popular cultivars of B. juncea, a susceptible (Kranti), and a
resistant (Bio-YSR) to act as watchdogs. All the thirty geno-
types were used to know the diverse disease reactions against
ten A. candida isolates collected from different major
mustard-growing areas of the country. Many of these geno-
types were unknown for white rust reactions to these collected
isolates. By the line selection, genotypes that have differenti-
ating potentialities were identified for white rust reaction and
the differentials were selected from these genotypes/cultivars.
Still most of them are unknown for the uniqueness or dupli-
cation of the white rust conditioning genes possessed by each
Indian mustard genotypes, it was invigorated from the work
done by Flor (1935). All the genotypes were grown under
controlled conditions and ten plants of each genotype were
inoculated with each isolate (separately) in two replications.
All the ten isolates of A. candida were inoculated in the same
set of conditions. Isolates of A. candida were designated by a
two-letter code (AC-code) followed by a hyphen and a listing
of those locations (short name) from where the isolate was
collected (Table 1). The statistically significant different iso-
lates of A. candida were categorized as physiological race of
A. candida and same were nominated based on the specific
host like- AcRBj-1 (Albugo candida Race B. juncea-1),
AcRBj-2 (Albugo candida Race B. juncea-2) and so on. An
Indian type-culture collection is proposed and will be main-
tained at the Division of Plant Pathology, ICAR-Indian
Agricultural Research Institute, New Delhi, India for further
use in breeding and research programs.
JPlantPathol
Statistical analysis
One-way ANOVA table (analysis of variance) was prepared
based on the differential reaction of thirty B. juncea genotypes
against ten Indian A. candida isolates to check whether there
were any statistical differences between the mean values
among the isolates. Further, the post hoc test, Tukey’s
Honest Significance Difference (Tukey’s HSD) test was used
to identify those A. candida isolates which are statistically
significantly different using the SPSS 20 software (Arbuckle
2011).
Results
Variations in size and shape of sporangia
Each isolate of A. candida collected from different locations
was observed to have a variable sporangial area, ranging from
199.01 μm
2
to 357.23 μm
2
with the largest being Ac-Bgpt
isolate while the smallest isolate being the Wb-Bbnr isolate
(Table 2, Fig. 1). The shape of sporangia was globular in Ac-
Bhrt, Ac-Bgpt, Ac-Mghy, Ac-Wltn, Ac-Cbr, Ac-Dli, Ac-Skn,
Ac-Kt, Ac-Gwlr, and Ac-Jpr isolates, whereas, it was spheri-
cal in Ac-Pnt and Ac-Kul isolates. Ac-Hr and Wb-Bbnr iso-
lates, on the other hand, had ellipsoidal shaped sporangia
(Table 2, Fig. 1).
Variations in size and shape of pustules
All the isolates showed creamy white color pustules except
Wb-Bbnr infecting A. blitum which has a glassy white appear-
ance. Different isolates of A. candida were found to have
varying sizes of pustules ranging from 0.5 mm to 3 mm in
diameter. The large size pustules, 1.5 mm to 3 mm were ob-
served in Ac-Bgpt, Ac-Hr, and Ac-Cbr isolates, and small to
medium size pustules were formed in the remaining isolates
(Table 2). Furthermore, different shapes of pustules were ob-
served on the infected leaves. Pinhead, raised, irregular, and
unevenly distributed pustules were observed in Ac-Bhrt, Ac-
Mghy and Ac-Gwlr isolates, while pinhead, raised, circular
pustules which were distributed uniformly all over the cotyle-
don were observed in Ac-Pnt, Ac-Wltn, Ac-Dli, Ac-Skn, Ac-
Kul, Ac-Kt, Wb-Bbnr, and Ac-Jpr isolates. Bigger size, raised
pustules arranged in a concentric ring having a pinhead type
dot in the center were observed in Ac-Bgpt, Ac-Hr, and Ac-
Cbr isolates (Supplementary Fig. 1).
Grouping of isolates based on qualitative traits
Based on three qualitative characters viz., the shape of pus-
tules and sporangia, and the color of pustules, the phenetic
dendrogram was constructed using 0–1 binary data through
NTSYS software. Fourteen isolates were clustered into four
distinct groups (Fig. 2). Ac-Bhrt, Ac-Mghy, Ac-Gwlr, Ac-
Bgpt, and Ac-Cbr isolates were clubbed in the first group with
more than 50% similarity. Cream color, pinhead shape, bigger
size pustules with irregular or concentric in shape, and globu-
lar shape sporangia were observed in these isolates. With
creamy color, pinhead size, and circular shape pustules (which
were uniformly distributed all over the cotyledons) with
spherical to globular sporangia, Ac-Pnt, Ac-Kul, Ac-Wltn,
Ac-Jpr, Ac-Dli, Ac-Kt and Ac-Skn isolates formed the second
group. Ac-Hr and Wb-Bbnr isolates, on the other hand, delin-
eated into a separate group. Ac-Hr isolate was having a sim-
ilarity of only 26% with the rest of the isolates, whereas, Wb-
Bbnr isolate expressed only 12% similarity to the rest of the
isolates. Both the isolates were delineated into different
groups because of the ellipsoidal shape of sporangia.
Grouping of isolates based on quantitative traits
To determine whether there were any significant differences
between the pustule diameter and sporangial area among dif-
ferent isolates of A. candida and W. bliti,multipleDuncan’s
range tests were performed. It was observed that based on the
sporangial area, all the isolates can be grouped into four clas-
ses (Table 2). Isolates viz., Wb-Bbnr, Ac-Pnt, Ac-Kul, Ac-
Gwlr, Ac-Jpr, and Ac-Cbr formed one group with the smallest
size sporangia. Isolates Ac-Kt, Ac-Wltn, Ac-Skn and Ac-Bhrt
formed the second group with small sporangia, however, big-
ger than the first group. Ac-Dli, Ac-Hr, and Ac-Mghy isolates
were represented in a separate group having medium size
sporangia. Ac-Bgpt isolates having the largest size sporangia
formed a separate group. Based on pustule diameter isolates
were divided using multiple Duncan’s range tests into three
different groups. It was observed that isolates viz., Ac-Dli, Ac-
Mghy, Ac-Jpr, and Ac-Kul were having small size pustules
and, therefore can be clubbed in one group. Whereas, Ac-
Bhrt, Ac-Pnt, Ac-Wltn, Ac-Kt, Ac-Gwlr, Ac-Skn, and Wb-
Bbnr isolates were having medium-sized pustules thus can be
clubbed into a separate group. With a larger size pustule, Ac-
Bgpt, Ac-Hr, and Ac-Cbr isolates formed a separate group
(Table 2).
PCA result
Based on the PCA analysis, five morphological characters
loaded on the first two principal components were found to
contribute about 65.3% of the total variability. PC1 accounts
for 37.7% of the total variability through the shape and size of
the sporangia and pustules while PC2 contributes 26.6% of
the total variability through pustule color and size of sporangia
and pustules (Fig. 3).
J Plant Pathol
Genetic diversity based on COX2 and ITS region
The evolutionary history was inferred by using the
Maximum Likelihood method based on the Tamura-Nei
model (Tamura and Nei 1993). Based on sequence analysis
of the COX2 region, thirteen A. candida isolates collected
from India showed a nucleotide identity range of 96.7–
99.4% with the other A. candida isolates used for the com-
parison from Europe, Asia, Africa, North America, and
Australia (Supplementary Fig. 2). A. candida isolates, Ac-
Bhrt, Ac-Bgpt, Ac-Mghy, Ac-Pnt, Ac-Hr, Ac-Cbr, and Ac-
Skn can be clubbed together in the first group with the Korean
A. candida isolate infecting B. juncea with a maximum nu-
cleotide similarity of 99.4% (Fig. 4). Ac-Jpr, Ac-Gwlr, Ac-
Dli, and Ac-Kul isolates formed a second group with a se-
quence similarity range of 97.1–98.8% with the rest of
A. candida isolates used for the comparison while Ac-Kt
and Ac-Wltn isolates form the third group with a lower se-
quence similarity range of 97.3–98.3% with the other
A. candida sequences from NCBI used for the comparison.
W. blilti collected from A. blitum is the most distant isolate
and formed a separated clade with A. amaranthi infecting
A. spinosus from Korea with the least sequence similarity
range of 79.4–82.6% compared with the rest of the
A. candida (Fig. 4). ITS sequence analysis of ten Indian
A. candida isolates infecting B. juncea revealed a nucleotide
identity range of 97.7–99.5% with the other A candida se-
quences from different continents used for the comparison
(Supplementary Fig. 3). Based on the maximum likelihood
phylogenetic tree, the A. candida isolates, Ac-Bhrt, Ac-Bgpt,
Ac-Mghy, Ac-Hr, and Ac-Cbr can be considered in a group
with the A. candida isolates from Korea and Australia col-
lected from B. juncea and B. oleracea var. gemmifera with a
nucleotide identity range of 98.3–99.3% (Fig. 5). Ac-Pnt,
and Ac-Skn isolates clubbed to form the second group while
Ac-Wltn, Ac-Jpr, and Ac-Dli isolates formed the third group
with a nucleotide identity range of 97.7–99 .3% with the other
A. candida isolates used for the comparison from the NCBI.
Table 2 Morphological characters among Indian isolates of A. candida used under this study
Isolate
code
Shape of pustules Color of
pustules
Pustule size
(mm)
Shape of
sporangia
Avg. sporangial size*
(μm
2
)
[π(0.5xW)(0.5xL)]*
Avg. pustule
diameter
(mm)**
Ac-Pnt Pin head, raised, circular pustule that scattered
all over leaf
Creamy white 0.5–2.0 Spherical 209.63
a
1.4
b
Ac-Kul Pin head, raised, circular pustule that scattered
all over leaf
Creamy white 0.5–1.5 Spherical 218.46
a
1.1
a
Ac-Skn Pin head, raised, circular pustule that scattered
all over leaf
Creamy white 1.0–2.0 Globular 260.40
b
1.6
b
Ac-Dli Pin head, raised, circular pustule that scattered
all over leaf
Creamy white 0.5–1.5 Globular 293.71
c
1.1
a
Ac-Jpr Pin head, raised, circular pustule that scattered
all over leaf
Creamy white 0.5–1.5 Globular 220.57
a
1.1
a
Ac-Wltn Pin head, raised, circular pustule that scattered
all over leaf
Creamy white 1.0–2.0 Globular 248.86
b
1.7
b
Ac-Kt Pin head, raised, circular pustule that scattered
all over leaf
Creamy white 0.5–2.0 Globular 247.34
b
1.5
b
Ac-Mghy Pin head, raised, irregular, unevenly
distributed pustule
Creamy white 0.5–1.0 Globular 315.43
c
0.9
a
Ac-Bhrt Pin head, raised, irregular, unevenly
distributed pustule
Creamy white 0.5–2.0 Globular 278.53
b
1.6
b
Ac-Gwlr Pin head, raised, irregular, unevenly
distributed pustule
Creamy white 1.5–2.0 Globular 238.36
a
1.6
b
Wb-Bbnr Pin head, raised, circular pustule Glassy white 1.0–2.0 Ellipsoid 199.01
a
1.7
b
Ac-Bgpt Bigger than pin head, raised pustule with one
concentric ring having pin head type dot in
centre
Creamy white 1.5–3.0 Globular 357.23
d
2.3
c
Ac-Hr Bigger than pin head, raised pustule with one
concentric ring having pin head type dot in
centre
Creamy white 2.0–3.0 Ellipsoidal 301.08
c
2.7
c
Ac-Cbr Bigger than pin head, raised pustule with one
concentric ring having pin head type dot in
centre
Creamy white 2.0–3.0 Globular 242.44
a
2.6
c
*W and L - average wide and length of the sporangia in μm
** Different letter indicates significant differences between the mean values using Duncan’s multiple range tests
JPlantPathol
Host differential reaction
Variability for disease development
Brassica genotypes when inoculated with different isolates of
A. candida observed variation for incubation period (IP),
which ranged from 3 to 9 days. Minimum IP (3 days) was
observed in Ac-Pnt isolates infecting JM-1, while maximum
IP (9 days) was recorded in Ac-Mghy isolate infecting PDZ-1
and Pusa Karishma. The latent period (LP), on the other hand,
varied from 7 to 12 days in the set of isolates taken for this
study. Most of the isolates took 9 days of LP across the geno-
types, however, a minimum latent period (7 days) was ob-
served in isolate Ac-Pnt, which was infecting the maximum
Fig. 1 Sporangial variability of
thirteen A. candida isolates
infecting B. juncea and one
W. bliti isolate infecting A. blitum
collected from different
geographical locations of India
observed under the light
microscope
Fig. 2 Dendrogram based on Jaccard’s similarity coefficients calculated from morphological traits recorded on fourteen isolates of Albugo species
J Plant Pathol
number of the host genotypes taken in this study. Maximum
LP (12 days) was recorded in Ac-Gwlr isolate while infecting
JM-1, EC 399299, and Kranti genotypes.
Host-pathogen interaction
All the thirty B. juncea genotypes used in the study were
able to give differential response against the ten Indian
A. candida isolates tested. The three genotypes, PDZ-3,
Pusa Karishma, and an internationally recognized resistant
genotype, Donskaja were found to give a resistance re-
sponse against all the ten isolates (Table 3). BioYSR, a
resistant genotype of B. juncea reported from India was
observed to show a susceptible reaction to Ac-Skn and
Ac-Wltn isolates while the PDZ-2 genotype was found to
be susceptible only against the Ac-Hr isolate. The remain-
ing genotypes expressed a mixed reaction of susceptibility
and resistance when inoculated against the ten isolates. Ac-
Skn, Ac-Hr, Ac-Gwlr, Ac-Jpr, and Ac-Bhrt were found to
be highly virulent as these isolates caused infections on a
total number of 24, 23, 21, 21, and 20 different B. juncea
genotypes with a higher disease severity whereas Ac-
Mghy isolate was found to be least virulent as it failed to
create disease symptoms in most of the tested lines except
only in nine genotypes where symptoms appeared with
very low disease severity (Table 3).
Post-hoc test analysis
Based on the ANOVA table, it was evident that statistically
significant differences were present between the mean values
of the disease reactions assay of different isolates against the
tested genotypes (Table 4). Further, to classify the A. candida
isolates, the post hoc test (Tukey’s HSD test) was performed
to exactly determine those isolates where statistically signifi-
cant differences were present. It was observed that the Ac-
Mghy isolate was statistically significantly different from the
rest of the A. candida isolates while Ac-Pnt, Ac-Bgpt isolates
were found to be significantly different from Ac-Skn, and Ac-
Hr isolates (Table 5). Whereas, no significant differences were
observed in the remaining isolates, viz. Ac-Bhrt, Ac-Gwlr,
Ac-Cbr, Ac-Jpr, and Ac-Wltn.
Discussion
Due to the wide host ranges, the bio-trophic pathogen
A. candida is highly diverse, and the presence of several bio-
logical races within the species complex has been suggested
previously (Choi et al. 2006,2007). There is an urgent need to
address this complexity in the pathogen and also to identify an
internationally standardized set of host differentials against
each host species. In this study, the degrees of variability
within the A. candida isolates from different geographical
Fig. 3 Biplot of different
variables loaded on PC1 and PC2
showing their contribution
towards total variation
JPlantPathol
regions of India were being investigated based on morpholog-
ical characters and molecular markers. Also, the nature of the
pathogenic reaction of such isolates was tested against a set of
different B. juncea lines.
Taxonomically important characters like shape and size of
sporangia and pustules, the color of pustules have been used
for studying morphological variability among thirteen
A. candida isolates and one W. bliti isolate. Most of the
sporangia were found to be globular to spherical except Wb-
Bbnr and Ac-Hr isolates which had ellipsoidal sporangia.
Varying sporangial sizes (sporangial area) were also observed
with a range between the smallest being 199.01 μm
2
and the
largest, 357.23 μm
2
. Globular to spherical sporangia were also
reported in the previous studies by Singh (1993),
Brandenberger et al. (1994), and Patni et al. (2005). Variable
size and shape of sporangia were reported independently by
Pounds and Williams (1963), Kolte (1985), and Lakra and
Saharan (1988) to differentiate the A. candida collections from
different host species. White rust pustules size and its distri-
bution patterns were reported to be used as a part of pathogen-
ic characters to classify the pathogen into different races
(Gupta and Saharan 2002). All the Indian isolates of
A. candida showed a white creamy colored pustule with a
diameter ranged between 0.5 mm - 3.0 mm. The biggest pus-
tule size was observed in Ac-Hr isolate (2.7 mm in dia.),
whereas, the smallest pustule was recorded in Ac-Mghy
(0.9 mm in dia.). Different morphological characters under
study were as valuable as good indicators of the variability
present among the pathogen isolates; however, these were not
substantial to classify them into races/pathotypes. Sequence
comparison of the two conserved regions of the pathogen i.e.
COX2 gene of mtDNA and ITS region of rDNA revealed a
nucleotide identity range of 96.7–99.5% when compared with
the retrieved sequences of other A. candida isolates reported
from different continents. Phylogenetic analysis showed that
all the Indian A. candida isolates collected from B. juncea
hosts belong to the group of A. candida sensu stricto reported
by Choi et al. (2006) with a close association with the Korean
isolates from B. juncea. All the A. candida isolates from dif-
ferent geographical origins of India can be classified into two
different groups based on a consensus phylogenetic tree anal-
ysis of COX2 and ITS sequence. The first group consists of the
isolates, Ac-Bhrt, Ac-Bgpt, Ac-Hr, Ac-Mghy, Ac-Pnt, Ac-
Skn, Ac-Cbr with identical sequences to each other (Figs. 4
and 5, Supplementary figs. 2and 3). The second group con-
sists of diverse isolates, Ac-Wltn, Ac-Kt, Ac-Kul, Ac-Dli, Ac-
Jpr, Ac-Gwlr with a lower sequence similarity range of 95.6–
98.8% to each other. So, there is a possibility of the presence
of more than one race within the group which requires further
experiment with the host differentials.
Fig. 4 Maximum likelihood
phylogenetic tree based on the
COX2 gene showing the
relationship of thirteen Indian
A. candida isolates from
B. juncea and one W. bliti isolate
from A. blitum compared with the
other A. candida isolates infecting
various hosts from different
continents
J Plant Pathol
Differential host plant interactions within a species are
believed to be the most reliable approach for differentiat-
ing pathogen variability and have been used for classify-
ing races/pathotypes in many crops (Levine and Stakman
1918; Roelfs and Martens 1988). Co-evolution of host
and pathogen under diverse selection pressure has led to
the creation of biological specialization in A. candida and
Brassica spp. including B. juncea. Moreover, A. candida
isolates collected from different Brassica spp. normally
showed to be most pathogenic on the host genotype or
species from where they originated (Minchinton et al.
2005), although they are also capable to infect heterolo-
gous hosts as well (Liu et al. 1996). Because of synteny in
the genome, individual isolates do not exhibit absolute
adaptation to one particular host species and, thus,
enforcing heterologous infection reactions (Tanhuanpaa
and Vilkki 1999). In Brassica spp., various isolates and/
or races have been classified based on their ability to
infect different hosts (Pound and Williams 1963;Lakra
and Saharan 1988,1989) or at least more than one species
of Brassicaceae (Rimmer et al. 2000) despite knowing
that isolates/races should be defined using a single host
species based on their differential reaction to the patho-
gen. This was primarily practiced due to the non-
availability of sufficient genetic diversity among the
B. juncea genotypes for white rust resistance. Once the
hosts interacting differently with different isolates of
A. candida are identified, it is imperative to establish
races based on B. juncea - A. candida interactions.
Thirty diverse B. juncea genotypes were taken to study
the differential reaction of ten A. candida isolates collect-
ed from the same host species (B. juncea) but from dif-
ferent regions and agro-climatic zones of India. As al-
ready available racial discrimination system on wheat
rustsinIndiaandtotheworld(StakmanandLevine
1922; Johnston and Mains 1932; Gassner and Straib
1932; Nagarajan et al. 1983), we have proposed a system
of thirty host differentials including Donskaja, a global
white rust resistant genotype along with the other
B. juncea genotypes variable to white rust disease. Our
set also contained popular cultivars such as Kranti (sus-
ceptible check), and Bio-YSR (resistant genotype) to act
as watchdogs. Although all the A. candida isolates under
study exhibited a differential response against the tested
B. juncea lines, based on Tukey’s HSD test, the ten iso-
lates were classified and grouped as four different physi-
ological races, designated as AcRBj1, AcRBj2, AcRBj3
andAcRBj4(Table6). Since all the isolates of A. candida
Fig. 5 Maximum likelihood
phylogenetic tree based on the
ITS region from ten Indian
A. candida isolates from
B. juncea compared with the other
A. candida isolates from different
continents.A.amaranthi
sequence retrieved from
GenBank was used as an
outgroup
JPlantPathol
were collected from different agro-climatic zones of India,
they have analyzed for morpho-molecular variations as
well as their virulence nature. All isolates gave a differ-
ential reaction response against a set of thirty B. juncea
genotypes tested that possess a set of resistance and
susceptibility genes, based on the significant difference
in disease reaction among the isolates, we have identified
four physiological races. These proposed races have qual-
ified all the criteria desired to became physiological races
like single host genetic diversity (a set of host differential)
Table 3 Differential reactions of thirty B. juncea genotypes against ten A. candida isolates collected from different geographical locations of India
Sl. No. B. juncea genotypes A. candida isolates
Ac-Bhrt Ac-Dli Ac-Hr Ac-Jpr Ac-Pnt Ac-Gwlr Ac-Skn Ac-Wltn Ac-Bgpt Ac-Mghy
1 Pusa Karishma 0 0 0 0 0 0 0 0 0 1
2PM22 6 36266 6 0 0 0
3 PM24 6 6 6 6 0 6 6 0 4 0
4 PM30 6 3 6 5 5 6 6 3 4 0
5PM29 6 06456 6 6 1 1
6 LES 49 0 0 0 6 0 5 6 6 5 1
7 LES 50 0 5 6 1 0 6 6 1 6 0
8 LES 51 6 0 6 6 0 0 6 0 6 0
9 PDZ-1 0 5 6 5 0 5 6 0 5 1
10 PDZ-2 0 0 6 0 0 0 0 0 0 0
11 PDZ-3 0 0 0 0 0 0 0 0 0 0
12 PDZ-4 5 4 6 6 0 6 0 0 5 1
13 RLC 3 6 0 6 3 0 0 6 0 0 0
14 EC 597318 6 6 6 6 0 0 0 6 5 1
15 JM-1 6 6 6 0 5 6 6 4 6 0
16 JM-2 6 6 6 6 6 6 6 5 6 0
17 JM-3 6 6 6 5 3 6 6 6 6 0
18 BioYSR 0 0 0 0 0 0 6 6 0 1
19 EC 399299 5 6 3 6 5 6 6 2 1 0
20 BEC 144 0 0 0 6 3 0 6 4 0 0
21 BEC 286 2 6 6 6 0 6 6 0 0 0
22 Donskaja 0 0 0 0 0 2 0 0 0 0
23 SEJ 8 6 0 5 6 1 6 6 6 0 0
24 RLM 619 6 6 6 6 6 6 6 3 4 0
25 Durgamani 5 6 6 6 6 4 6 2 1 0
26 Rohini 2 6 6 6 5 6 6 6 5 0
27 Pusa Vijay 5 6 6 6 5 6 6 6 5 0
28 RL 1359 6 0 6 6 5 6 6 6 0 0
29 Kranti 4 6 6 6 4 6 6 5 0 1
30 EC 399299 6 6 6 6 0 6 6 4 1 1
*The disease rating scale of 0–6 was used at the cotyledonary stage (Conn et al. 1990)
Table 4 One way ANOVA table for the ten independent treatments
Source Sum squares (SS) Degree of freedom (ν) Mean square (MS) F statistic p value
Between groups 496.6033 9 55.1781 9.1169 3.74E-12
Within groups 1755.17 290 6.0523
Total 2251.77 299
J Plant Pathol
as well as diversity among pathogen isolates, therefore, it
can be considered as a novel and different races (Flor,
1935, Nagarajan et al. 1983). It is very much unalike from
the previously reported 49 races in India which was not
assessed on single host genetic diversity with statistical
analysis and also they were based on pathogenic reaction
to multiple host species (Singh and Bhardwaj 1984;Lakra
and Saharan 1988; Bhardwaj and Sud 1988; Verma et al.
1999;Jat1999; Gupta and Saharan 2002).
The host differentials, taken in this study, are expected
to possess different gene(s) or set of genes, which can be
identified and deployed into a single nuclear background,
to develop near-isogenic lines (NILs), for their further use
in the development of a set of host differentials. Until
then, identified resistance sources can be deployed for
the development of the region or agro-climatic zone-wise
specific cultivar possessing durable resistance. The mate-
rial and information emanating from this study will help
in understanding the variability within A. candida,identi-
fication of new genes imparting resistance against this
pathogen, and their deployment for management of white
rust disease through the development of mustard
(B. juncea) cultivars with durable and/or wide-spectrum
resistance.
Table 5 Tukey’s HSD test results showing the p-values to validate the pairwise statistical differences between the isolates
Isolates code Ac-Dli Ac-Hr Ac-Jpr Ac-Pnt Ac-Gwlr Ac-Skn Ac-Wltn Ac-Bgpt Ac-Mghy
Ac-Bhrt 0.900
(NS)
0.900
(NS)
0.900
(NS)
0.459
(NS)
0.900
(NS)
0.781
(NS)
0.900
(NS)
0.653
(NS)
0.001
(S**)
Ac-Dli 0.459
(NS)
0.877
(NS)
0.900
(NS)
0.900
(NS)
0.321
(NS)
0.900
(NS)
0.900
(NS)
0.001
(S**)
Ac-Hr 0.900
(NS)
0.011
(S*)
0.900
(NS)
0.900
(NS)
0.148
(NS)
0.030
(S*)
0.001
(S**)
Ac-Jpr 0.087
(NS)
0.900
(NS)
0.900
(NS)
0.525
(NS)
0.188
(NS)
0.001
(S**)
Ac-Pnt 0.130
(NS)
0.005
(S**)
0.900
(NS)
0.900
(NS)
0.048
(S*)
Ac-Gwlr 0.900
(NS)
0.621
(NS)
0.263 0.001
(S**)
Ac-Skn 0.087
(NS)
0.015
(S*)
0.001
(S**)
Ac-Wltn 0.900
(NS)
0.002
(S**)
Ac-Bgpt 0.018
(S*)
NS, Statistically non-significant; S, Statistical significant
*Statistically significant at p<0.05
**Statistically significant at p<0.01
Table 6 Delineation of collected Indian A. candida isolates into different physiological races based on the Tukey’s HSD test
Sl. No Isolates code Isolates with significant differences observed in the Tukey HSD test result Proposed race
1. Ac-Mghy Ac-Hr, Ac-Pnt, Ac-Skn, Ac-Bgpt, Ac-Bhrt, Ac- Dli, Ac-Jpr, Ac-Gwlr, and Ac-Wltn AcRBj1
2. Ac-Pnt Ac-Mghy, Ac-Hr, and Ac-Skn AcRBj2
3. Ac-Bgpt
4. Ac-Hr Ac-Mghy, Ac-Pnt, and Ac-Bgpt AcRBj3
5. Ac-Skn
6. Ac-Bhrt Ac-Mghy AcRBj4
7. Ac- Dli
8. Ac-Jpr
9. Ac-Wltn
10. Ac-Gwlr
JPlantPathol
Acknowledgments The authors are thankful to the Director, ICAR-IARI,
Pusa, New Delhi-12, India, for providing financial assistance to carry out
this research work.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent Not applicable.
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