Genetic diversity and population structure of ‘Candidatus Liberibacter asiaticus’ associated with citrus Huanglongbing in India based on the prophage types

Abstract

Huanglongbing (HLB), also known as ‘citrus greening’, is an extremely destructive disease of citrus worldwide. HLB is associated with three species of the fastidious proteobacterium, Candidatus Liberibacter asiaticus (CaLas), Ca. L. africanus and Ca. L. americanus with CaLas being the most widely distributed around the world and the only species detected and described so far in India, one of the major global citrus fruit producers. Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. Three types of prophages, Type 1, Type 2 and Type 3 have been identified and described in CaLas so far. In the present study, 441 CaLas isolates sampled across 18 Indian states were used for prophage typing. Based on detection of three prophage types by PCR, all the eight probable combinations of CaLas prophages were identified, including single Type 1 (26.5%), single Type 2 (18.8%), single Type 3 (1.4%), Type 1 + Type 2 (20.4%), Type 1 + Type 3 (12.5%), Type 2 + Type 3 (4.8%), Type 1 + Type 2 + Type 3 (11.3%) and None type (4.3%). Prophage types were confirmed by PCR amplicon sequencing and subsequent phylogenetic analysis. By discovery of all 3 prophages and based on genetic identity and genetic distance, CaLas populations from eighteen citrus growing states were separated into two major Prophage Typing Groups (PTGs): PTG1 and PTG2. The PTG1 comprised of CaLas from North-West India and PTG2 from rest of the country (North-East, Central and South India), and both major groups were further divided into two (PTG1-A, PTG1-B) and three (PTG2-A, PTG2-B and PTG2-C) subgroups respectively. The findings of CaLas population patterns provide evidence for independent origins of HLB-associated CaLas. CRISPR (clustered regularly interspaced short palindromic repeats) array was also detected in CaLas isolates. This is the first report evaluating the genetic variation of a large population of CaLas bacterium in India using the PCR markers from the prophage regions which would certainly assist the ongoing HLB management efforts in India.

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World Journal of Microbiology and Biotechnology (2021) 37:95
https://doi.org/10.1007/s11274-021-03057-8
ORIGINAL PAPER
Genetic diversity andpopulation structure of‘Candidatus Liberibacter
asiaticus’ associated withcitrus Huanglongbing inIndia based
ontheprophage types
AshisK.Das1· SubhamA.Chichghare1· SusheelK.Sharma2· J.PrasanthTejKumar1· SalvinderSingh3·
VirendraK.Baranwal4· AshokKumar1· SagarNerkar1
Received: 26 January 2021 / Accepted: 19 April 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021
Abstract
Huanglongbing (HLB), also known as ‘citrus greening’, is an extremely destructive disease of citrus worldwide. HLB is
associated with three species of the fastidious proteobacterium, Candidatus Liberibacter asiaticus (CaLas), Ca. L. africanus
and Ca. L. americanus with CaLas being the most widely distributed around the world and the only species detected and
described so far in India, one of the major global citrus fruit producers. Prophages are highly dynamic components in the
bacterial genome and play an important role in intraspecies variations. Three types of prophages, Type 1, Type 2 and Type 3
have been identified and described in CaLas so far. In the present study, 441 CaLas isolates sampled across 18 Indian states
were used for prophage typing. Based on detection of three prophage types by PCR, all the eight probable combinations
of CaLas prophages were identified, including single Type 1 (26.5%), single Type 2 (18.8%), single Type 3 (1.4%), Type
1 + Type 2 (20.4%), Type 1 + Type 3 (12.5%), Type 2 + Type 3 (4.8%), Type 1 + Type 2 + Type 3 (11.3%) and None type
(4.3%). Prophage types were confirmed by PCR amplicon sequencing and subsequent phylogenetic analysis. By discovery
of all 3 prophages and based on genetic identity and genetic distance, CaLas populations from eighteen citrus growing states
were separated into two major Prophage Typing Groups (PTGs): PTG1 and PTG2. The PTG1 comprised of CaLas from
North-West India and PTG2 from rest of the country (North-East, Central and South India), and both major groups were
further divided into two (PTG1-A, PTG1-B) and three (PTG2-A, PTG2-B and PTG2-C) subgroups respectively. The find-
ings of CaLas population patterns provide evidence for independent origins of HLB-associated CaLas. CRISPR (clustered
regularly interspaced short palindromic repeats) array was also detected in CaLas isolates. This is the first report evaluating
the genetic variation of a large population of CaLas bacterium in India using the PCR markers from the prophage regions
which would certainly assist the ongoing HLB management efforts in India.
Keywords “Candidatus Liberibacter asiaticus”· Citrus· Genetic diversity· Huanglongbing· Prophage
Introduction
Huanglongbing (Chinese name for yellow dragon disease or
yellow shoot disease, abbreviated as HLB), aka citrus green-
ing, is the most ruinous disease of citrus all over the globe
as of today (da Graca etal. 2016, Wang etal. 2017). A cen-
tury-old problem in citrus cultivation (Bove 2006), currently
the disease has spread to more than 50 countries in Africa,
Asia, Oceania, and the Americas (South, North and the Car-
ibbean) (Dala-Paula etal. 2019). HLB is associated with
three nonculturable Gram-negative, α-proteobacterial spe-
cies (of the Rhizobiaceae family), ‘Candidatus Liberibac-
ter asiaticus’ (CaLas), ‘Ca. Liberibacter africanus’ (CaLaf)
(Jagoueix etal. 1994) and ‘Ca. Liberibacter americanus’
* Ashis K. Das
dasashiskumar@hotmail.com
1 Plant Pathology Lab, ICAR-Central Citrus Research
Institute, Amravati Road, Nagpur440033, India
2 ICAR Research Complex forNEH Region, Manipur Centre,
Imphal795004, India
3 Department ofAgricultural Biotechnology, Assam
Agricultural University, Jorhat785013, India
4 Advanced Centre forPlant Virology, ICAR-Indian
Agricultural Research Institute, NewDelhi110012, India
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World Journal of Microbiology and Biotechnology (2021) 37:95
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(CaLam; Teixeira etal. 2005). Within infected citrus plants,
Liberibacter spp. strictly resides in phloem sieve cells; and
has a small genome of approximately ~ 1.25 Mb (Duan etal.
2009). CaLas and CaLam are transmitted by the Asian citrus
psyllid (Diaphorina citri) and CaLaf is transmitted by the
African citrus psyllid (Trioza erytreae) apart from spread
by grafting during vegetative propagation (Bove 2006).
Among the three species, CaLas is the most widespread spe-
cies found in Asia and North America, and recently in East-
ern Africa (Ajene etal. 2020). In South America, though
both CaLam and CaLas are present, in recent times CaLas
has nearly abolished CaLam to become the most dominant
species there (Lopes etal. 2009). No effective and durable
control for HLB is available till date. Billions of dollars of
annual economic loss is related to yield, quality and tree
decline, and recurring expenditures for control and regula-
tion measures of HLB (Bove 2006; Gottwald 2010; Li etal.
2020).
The presence of HLB in India was first described dur-
ing 1960s (Fraser etal. 1966; Fraser and Singh 1968). The
disease was initially thought to be caused by a virus, as fas-
tidious prokaryotes (then known as Mycoplasma-like organ-
isms, MLOs and bacterium-like organism, BLOs) were not
known at that time. Subsequently, the so called "greening
virus" was found transmitted by the psyllid vector, D. citri
(Capoor etal. 1967). Occurrence of HLB was later reported
from different parts of the country (Varma etal. 1993; Das
and Singh, 1999). Conventional PCR and real time quan-
titative PCR (qPCR)- based molecular identification of
CaLas was reported and substantiated from Central India
and North-Eastern India later (Das etal. 2007, 2014; Das
2009). CaLas infects all cultivated citrus varieties in India
and causes various symptoms on citrus plants, mainly on the
canopies and leaves. The typical symptoms of HLB-affected
citrus include yellow shoot, asymmetric blotchy-mottle on
leaves, and inward leaf curl with vein corking. HLB-affected
trees may be smaller with upright leaves, have lopsided, bit-
ter fruit, exhibits early leaf-drop and die-back, and often die
in 3–8 years after becoming symptomatic. However, many
HLB-affected trees do not show uniform symptoms and may
have branches that are free of all symptoms (Bove 2006). It
was hypothesized that the spatial and temporal variations of
different CaLas populations may contribute to the variations
of bacterial titers and HLB symptom expression observed in
the infected host plants (Zhou etal. 2013).
Our previous survey recorded upto 47% HLB incidence
in different commercial citrus cultivars in India (Das 2008)
and nearly 30–40% crop loss has been recorded recently in
Nagpur mandarin (C. reticulata) production belts of Central
India. Symptom variations on HLB-affected citrus plants
were found to be associated with the specific CaLas popu-
lations in Florida based on the genetic diversity within the
CaLas prophage regions (Zhou etal. 2013). However, there
is only little information regarding the distribution of genetic
diversity of CaLas in India.
Before the availability of the complete CaLas genome
sequence information data (Duan etal. 2009), the detec-
tion, identification and genetic diversity studies of CaLas
bacteria were limited to the sequences of a few housekeep-
ing genes, such as the 16S rRNA gene, 16S/23S rRNA
intergenic spacer regions (Jagoueix etal. 1994, 1997; Li
etal. 2006; Ding etal. 2009), the outer membrane protein
(omp) gene (Bastianel etal. 2005) or the β-operon gene loci
(Teixeira etal. 2008; Villechanoux etal. 1993). However,
genetic variation within these conserved genes has limited
discriminatory power to differentiate closely-related iso-
lates within CaLas populations. Another chromosomal gene
deoxy-ribonucleotide reductase gene (nrdB) has also been
used for CaLas species identification (Zheng etal. 2016b). A
genomic locus, trn with short tandem repeats, was found to
be highly variable and has been used in population diversity
studies (Chen etal. 2010; Katoh etal. 2011). The resulting
diversity studies using these housekeeping genes revealed
restricted variation amongst the CaLas global isolates. Since
the publication of the CaLas genome sequences, a number
of loci, especially repetitive sequences/tandem repeats and
prophage regions, have been shown to be superior markers
for differentiating CaLas isolates from different geographical
origins (Liu etal. 2011; Zhou etal. 2011; Islam etal. 2012;
Puttamuk etal. 2014; Fu etal. 2020).
Bacteriophages or, Phages are the most abundant organ-
isms in the earth’s biosphere with an estimated 1031 parti-
cles (Keen 2015; Mushegian 2020). After invading a living
bacterial cell, bacteriophages can multiply using bacterial
materials and phage enzymes by two different mechanisms,
the lytic cycle as phage and the lysogenic cycle as prophage.
Prophages, the lysogenic form of a bacterial phage with its
DNA inserted into the chromosome, are important genetic
elements of the bacterial genome and play key roles in bac-
terial evolution, bacterial cell defense, and environmental
adaptation including pathogenesis (Boyd and Brüssow
2002; Feiner etal. 2015). In the lysogeny cycle, temperate
phages can participate in horizontal gene transfer (Touchon
etal. 2017) and regulate bacterial host behavior throughout
lysogenic conversion and interactions between them (Argov
etal. 2017). It is also reported that the presence of prophages
can increase bacterial fitness and ecological competitiveness
(Abedon 2008; Fortier and Sekulovic 2013) and could pro-
mote the adaptive evolution of bacterial pathogens (Davies
etal. 2016; Weitz etal. 2017). Therefore, the impact of tem-
perate phages on their hosts could be manyfold, and interac-
tions between them are enormously complex.
In CaLas, three types of prophages, Type 1 (SC-1 type),
Type 2 (SC-2 type), and Type 3 (P-JXGC-3), have been
described (Zhang etal. 2011; Zheng etal. 2016a, 2018),
based on currently available sequence data. Available
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World Journal of Microbiology and Biotechnology (2021) 37:95
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research findings suggested that SC1 was involved in the
lytic cycle of forming phage particles and SC2 was involved
in the lysogenic conversion of CaLas pathogenesis (Zhang
etal. 2011; Fleites etal. 2014; Jain etal. 2015). A CRISPR
(clustered regularly interspaced short palindromic repeats)/
cas system was reported in all the prophages which was
predicted to be involved in bacterial defense by cleaving
invading phage DNA based on spacer sequences (Zheng
etal. 2016a). The Type 3 phage was reported to carry a
unique bacterial defense system, called restriction-modifi-
cation system (Zheng etal. 2018). Different combinations
of above prophages have also been identified in CaLas
samples (Zheng etal. 2018). A new Type 4 prophage-like
sequence has also been described recently (Dominguez-
Mirazo etal. 2019). Several characteristics suggested that
Type 4 sequences were remnants of a prophage integrated in
the bacterial genome. Phage particles were observed in the
experimental host periwinkle and in infected citrus (Zhang
etal. 2011; Fleites etal. 2014; Fu etal. 2015).
The diversity and genetic structure within a given patho-
gen population can be a valuable resource for determining
the source or origin of the pathogen and risk management of
the disease to which it is associated with. Previous studies of
CaLas diversity in different regions of India were carried out
based on tandem repeat numbers in the CLIBASIA_01645
locus (Ghosh etal. 2015; Singh etal. 2019) but no prophage
region-based research was conducted so far. In the present
investigation, CaLas isolates collected from eighteen cit-
rus growing states were studied and analysed for genetic
diversity largely focusing on their prophage regions. Three
different prophage types along with eight prophage combina-
tions were detected and different prophage typing popula-
tion groups and subgroups were identified, described and
discussed in the context of probable geographical origin of
the HLB-associated CaLas bacterium. CRISPR repeat ele-
ments were also detected and identified in prophage region
of different CaLas isolates from India.
Materials andmethods
HLB sample collection
Citrus samples were collected through a number of sur-
veys from 2014 through 2019 in 18 states of India, namely
Punjab, Rajasthan and Gujarat in North-West (NW) India,
Madhya Pradesh and Maharashtra in Central India, Andhra
Pradesh, Telangana, Karnataka and Tamil Nadu in South-
ern India, and the nine states in North-Eastern (NE) India
(Arunachal Pradesh, Assam, Meghalaya, Manipur, Mizo-
ram, Nagaland, Tripura, Sikkim and sub-Himalayan tracts
of West Bengal) (Fig.1). Different citrus varieties/cul-
tivars like sweet orange (Mosambi, Sathgudi, Valencia),
mandarin (Nagpur, Kinnow, Coorg, Khasi, Sikkim, Dar-
jeeling, Daisy, Michal), acid lime (Kaghzi, Pramalini, Jay-
adevi) and lemon (Assam, Lisbon, Elachi), Pomelo, Gol
nimbu, Memang Narang, citron, Satkara and rootstock spe-
cies (Rough lemon, Rangpur lime) were sampled. Details
of citrus samples collected are elaborated in Supplemen-
tary TableS1. Most of these varieties are commercial cit-
rus species in different parts of India (TableS1).
Citrus tissue samples (n = 1018) were acquired from
the citrus trees showing HLB-like symptoms consisting
of classic blotchy mottling and/or chlorosis of leaves; yel-
lowish shoots; vein corking; zinc-like deficiency; small,
asymmetrical, poorly coloured fruit, aborted seeds, fruits
showing lopsidedness, oblong stylar end greening (Fig.2).
In case of leaves, fully expanded leaves from symptomatic
branches were sampled. Samples were taken from trees,
put in re-sealable plastic zip-lock bags with wet paper
cloths and immediately shipped to the ICAR- Central
Citrus Research Institute, Nagpur, India. Each sample or
isolate represented a single different tree and CaLas DNA
was extracted from citrus tissues as described below.
Fig. 1 The surveyed citrus growing states of India. PB Punjab, RJ
Rajasthan, GJ Gujarat, MP Madhya Pradesh, MH Maharashtra, AP
Andhra Pradesh, KA Karnataka, TN Tamil Nadu, SK Sikkim, ML
Meghalaya, TR Tripura, AR Arunachal Pradesh, TG Telangana, AS
Assam, NL Nagaland, MN Manipur, MZ Mizoram and WB West Ben-
gal
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DNA extractions andCaLas detection bycPCR
andqPCR
About 0.2 g of mid rib/petioles from leaf or, 0.2 g of central
column (vascular tissues) from fruit samples were taken and
crushed in liquid nitrogen using mortar and pestle and the
ground samples were used for the isolation of DNA using
DNeasyR Plant Mini Kit (Qiagen,Valencia, CA, USA) as
per the manufacturer’s protocol. In the final step, DNA
samples were eluted in 100 μl of buffer AE (Qiagen) and
stored at − 20 °C until further use. DNA concentrations of
all samples were measured individually with a NanoVue plus
spectrophotometer (GE Life Sciences, Marlborough, MA,
USA). Infection of CaLas was confirmed in the extracted
DNA by the real time quantitative PCR (qPCR) procedure
described by Li etal. 2006. TaqMan qPCR amplifications
were performed in a ABI 7300 (Applied Biosystems, Foster
City, Calif., USA) machine using specific primers HLBas,
HLBr and probe HLBp (Table1). The 15 µl TaqMan PCR
reaction mixture contained 7.5 µl of TaqMan PCR master
mix (Applied Biosystems), 250 nM of each primer, 150 nM
of probe HLBp, and 100 ng of DNA template. The real-time
Table 1 Prophage type specific primer sets and 16S rDNA specific primer sets used in this study
a Amplicon size
b Temperature at which annealing stage was carried out during PCR
c hsd for host specificity determinant
Name Sequence 5′-3′Size (bp)aTarget region Specificity Temp (°C)bReference
T1-2F TGG CTC GGG TTC AGG
TAA AT
975 Endolysin Type 1 prophage 60 (Zheng etal. 2016a)
T1-2R AAG GGC GAC GCA TGT
ATT TC
T1-3F CTC ACT GCG TCT TGA
TTC GG
866 Hypothetical protein gene Type 1 prophage 60 (Zheng etal. 2016a)
T1-3R CGA ACG AGC GGT ATG
TTT GT
T2-2F ACC CTC GCA CCA TCA
TGT TA
813 Phage structural protein
gene
Type 2 prophage 60 (Zheng etal. 2016a)
T2-2R TCG TCT TGA TTG GGC
AGA GT
T2-3F ACA GTT AAG AGC CAC
GGT GA
918 Hypothetical protein gene Type 2 prophage 60 (Zheng etal. 2016a)
T2-3R AAG ACG TGG GTG TTA
TGG GT
891-1F CTG ATC CTT TAC CAT
GCC GC
950 hsdScType 3 prophage 60 (Zheng etal. 2018)
891-1R CAG CGA AAC CGA TCT
TGA GG
891-2F ACC GCG ATC TAC CCG
TAA TT
884 hsdRcType 3 prophage 60 (Zheng etal. 2018)
891-2R TGT GTT TTG CGA GTG
AAG GG
CRIF CTC AGC TTT TGT CAT
GCC CA
~ 500 CRISPR region CaLas prophage region 56 (Zheng etal. 2016a)
CRIR AGG AAG ACA ATA TCG
CCC GT
Las606 GGA GAG GTG AGT GGA
ATT CCGA
500 16S rRNA gene CaLas chromosomal
region
58 (Fujikawa and Iwanami,
2012)
LSS ACC CAA CAT CTA GGT
AAA AACC
HLBas TCG AGC GCG TAT GCA
ATA CG
75 16S rRNA gene CaLas chromosomal
region
58 (Li etal. 2006)
HLBar GCG TTA TCC CGT AGA
AAA AGG TAG
HLBp FAM- AGA CGG GTG
AGT AAC GCG -BHQ1
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World Journal of Microbiology and Biotechnology (2021) 37:95
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PCR amplification setting included 95 °C denaturation for
15 min, followed by 40 cycles of 94 °C for 15 s and 58 °C
for 60 s. Cycle threshold (Ct) values based on fluorescent
signals were determined using Applied Biosystems 7300
Real-Time PCR System Software SDS version1.4.1. Sam-
ples were considered qPCR-positive if the Ct (cycle thresh-
old) value was less than 32 (Das etal. 2014) and selected for
further prophage detection.
The Presence of CaLas was also confirmed by conven-
tional PCR (cPCR) with primer set Las606/LSS (Fujikawa
and Iwanami 2012). DNA amplification was conducted in
a total volume of 25 μl using DreamTaq DNA polymer-
ase (Thermo Fisher Scientific Inc., MA, USA). PCR was
performed on a Bio-Rad T100™ Thermal Cycler (Bio-Rad,
Hercules, CA, USA) under the following procedure: initial
denaturation at 96 °C for 3 min, 35 cycles of amplification
(94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min) and
a final extension of 72 °C for 10 min. The PCR products
were electrophoresed in 1.5% agarose gel (1 X TAE buffer)
and visualized by Biovis Gel Doc System (Mumbai, India)
under UV illumination. Some of the representative PCR
products were directly sequenced by outsourcing (Euro-
fins, Bangalore, India) and sequences obtained were further
analyzed using BLAST (http:// blast. ncbi. nlm. nih. gov/ Blast.
cgi?) to verify the species identity. All the positive DNA
samples were designated as CaLas isolates.
Fig. 2 Symptoms associated with Huanglongbing (citrus greening
disease) in different citrus cultivars grown in different states of India.
Appearance of ‘yellow shoot’ in Nagpur mandarin in Maharashtra
(a). Classic ‘blotchy mottle’ leaf symptom in: Khasi mandarin in
Arunachal Pradesh (b) Sathgudi sweet orange in Andhra Pradesh (c),
C. indica in Meghalaya (d), Mosambi in Maharashtra (e), Gol nimbu
in Assam (f) Nagpur mandarin in Maharashtra (g), Assam lemon in
Assam (h), Lisbon lemon in Nagaland (i). Fruit symptoms: Kinnow
mandarin fruit showing stylar end greening in Punjab (j), stylar end
greening of Valencia fruits in Maharashtra (k), Lopsided Sathgudi
fruit showing blotchy mottle on fruit surface in Andhra Pradesh (l),
Nagpur mandarin fruit showing aborted seed in Maharashtra (m), and
lopsided pink-fleshed Pomelo fruit in Gujarat state; Note the non-col-
oration of the flesh in the affected portion of the fruit (n)
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World Journal of Microbiology and Biotechnology (2021) 37:95
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Amplification ofprophage region
All CaLas isolates were subjected to prophage-typing
using six primer sets described previously (Zheng etal.
2016b, 2018). Primer sets T1-2F/T1-2R and T1-3F/T1-3R
specific to prophage Type 1, T2-2F/T2-2R and T2-3F/
T2-3R specific to prophage Type 2 and 891-1F/891-1R and
891-2F/891-2R specific for prophage Type 3 were used for
PCR amplification of the prophage regions. A schematic
diagram presenting the arrangement of prophage regions
alongwith the locations of various type-specific primers
is illustrated in Supplementary Fig. S1. All primers used
in this study are detailed in Table1 and were synthesized
by Integrated DNA Technologies, Inc. (Coralville, IA,
USA). Standard conventional PCR was performed on a
Bio-Rad T100™ Thermal Cycler (Bio-Rad, Hercules, CA,
USA). The 25 μl reaction mixture containing 1 μl of DNA
template (approximate 100 ng), 0.2 μl of Dream Taq DNA
polymerase at 5 U/μl, 0.4 μl of 10 mM deoxynucleotide
triphosphates (dNTPs), 2.5 μl of 10 × DNA polymerase
buffer with MgCl2, 0.5 μl of each forward and reverse
primer (10 μM) and 19.9 μl of ddH2O. PCR was performed
under the following procedure: initial denaturation at 96
°C for 3 min, 35 cycles of amplification (94 °C for 30 s,
60 °C for 30 s, and 72 °C for 60 s) and ended with a final
extension of 72 °C for 10 min. The PCR products were
electrophoresed in 1.2% agarose gels (1 × TAE buffer)
containing ethidium bromide (0.5 µg/ml) and visualized/
photographed by Biovis Gel Doc System (Mumbai, India)
under UV illumination.
All the CaLas isolates were then classified into eight
possible prophage-based groups: Type 1, Type 1 + Type 2,
Type 1 + Type 3, Type 1 + Type 2 + Type 3, Type 2, Type
2 + Type 3, Type 3 and no prophage.
Table 2 Frequencies of different prophage combinations in “Candidatus Liberibacter asiaticus”-infected citrus DNA samples in 18 different
states of India as determined by Type specific primer sets
a All CaLas strains are positive for CaLas infection using qPCR method of Li etal (2006) and cPCR using Las606/Lss (Fujikawa and Iwanami
2012) primers
b Distribution of different prophage combinations in “Candidatus Liberibacter asiaticus”-infected citrus DNA samples determined by Type spe-
cific primer sets described in Table1
c Figures in parentheses are percentage frequency
STATE No. of isolatea# CaLas samples with prophage combinationb (frequency, %)
Type 1 Type 2 Type 1 + Type
2
Type 2 + Type
3
Type 1 + Type
3
Type
1 + Type
2 + Type 3
Type 3 Neither Type
1 + Type
2 + Type 3
Andhra
Pradesh
19 5 (26.3)c1 (5.3) 2 (10.5) 4 (21.1) 3 (15.8) 2 (10.5) – 2 (10.5)
Arunachal
Pradesh
11 3 (27.3) 2 (18.2) 3 (27.3) – 1 (9.1) 2 (17.2) – –
Assam 57 20 (35.1) 1 (1.8) 30 (52.6) – 3 (5.3) 3 (5.3) – –
Gujarat 10 – 10 (100.0) – – – – – –
Karnataka 24 4 (16.7) 6 (25.0) 1 (4.2) 2 (8.3) 2 (8.3) 7 (29.2) 1 (4.2) 1 (4.2)
Madhya
Pradesh
68 8 (11.8) 10 (14.7) 4 (5.9) 4 (5.9) 23 (33.8) 15 (22.1) 1 (1.5) 3 (4.4)
Maharashtra 69 22 (31.9) 11 (15.9) 10 (14.5) 8 (11.6) 8 (11.6) 7 (10.1) 1 (1.4) 2 (2.9)
Manipur 9 3 (33.3) 2 (22.2) 2 (22.2) – 1 (11.1) 1 (11.1) – –
Meghalaya 17 2 (11.8) 2 (11.8) 11 (64.7) – 1 (5.9) 1 (5.9) – –
Mizoram 15 5 (33.3) 4 (23.5) 4 (23.5) – 1 (6.7) 1 (6.7) – –
Nagaland 12 6 (50.0) 2 (16.7) 1(8.3) – 1 (8.3) 2 (16.7) – –
Punjab 39 8 (20.5) 19 (48.7) 6 (15.4) – – – – 6 (15.4)
Rajasthan 16 3 (18.8) 7 (43.8) 3 (18.8) – – – – 3 (18.8)
Sikkim 12 3 (25.0) 2 (16.7) 3 (25.0) – 2 (16.7) 2 (16.7)
Tamilnadu 14 9 (64.3) – – – 2 (14.3) – 2 (14.3) 1 (7.1)
Telangana 15 4 (26.7) 1 (6.7) 2 (13.3) 3 (20.00) 2 (13.3) 1 (6.7) 1 (6.7) 1 (6.7)
Tripura 11 2 (18.2) 1 (9.1) 3 (27.3) – 2 (18.2) 3 (27.3) – –
West Bengal 23 10 (43.5) 2 (8.7) 5 (21.7) – 3 (13.0) 3 (13.0) – –
Total 441 117 (26.5) 83 (18.8) 90 (20.4) 21 (4.8) 55 (12.5) 50 (11.3) 6 (1.4) 19 (4.3)
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World Journal of Microbiology and Biotechnology (2021) 37:95
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Sequencing ofType 1, Type 2 andType 3 prophages
andphylogenetic analyses
Seven numbers each of T1-2F/T1-2R and T2-2F/T2-2R
amplicons and Four numbers of 891-F/891-R PCR ampli-
cons were purified using QIAquick PCR purification kit
(Qiagen, Valencia, CA, USA) and directly sequenced
in both directions through a commercial DNA sequenc-
ing service (Eurofins, Bangalore, India). The consensus
sequence from the assembled contigs for each individual
isolate was subjected to an NCBI BLAST search (http://
www. ncbi. lm. nih. gov/ BLAST/) in the GenBank database.
The obtained 18 Prophage sequences from this study
and 9 sequences downloaded from GenBank database
were aligned through CLUSTAL package (Thompson etal.
1994) and were embedded and analyzed using MEGA X
software (Kumar etal. 2018) through neighbour—joining
distance methods and the phylogenic tree was constructed
using the maximum composite likelihood method (Tamura
etal. 2004) with 1000 bootstrap replicates.
Genetic data analysis ofCaLas populations
Analysis of genetic diversity of CaLas populations was
carried out using POPGENE version 1.32 software (Yeh
etal. 1999) (https:// sites. ualbe rta. ca/ ~fyeh/ popge ne. html).
Analysis procedures were followed as described previously
(Li etal. 2019). Briefly, as elaborated before, 6 pairs of
primers (2 pairs of primers for each prophage type) were
used. The positive amplification (presence) was marked
as “1” and no amplification (absence) was marked as “2”
and recorded in an MS Excel worksheet as per the data
input requirement of POPGENE v 1.32 and analysed in
Haploid mode. Subsequently, a pairwise matrix of genetic
distance and genetic identity for CaLas strains was gener-
ated with the same POPGENE software using Nei’s unbi-
ased measure and Nei’s (1973) gene diversity index (H
value) was estimated for CaLas populations in association
with prophages and individual citrus cultivating states of
India. The gene diversity of a locus, also known as its
expected heterozygosity (H) describes the proportion of
heterozygous genotypes expected under Hardy–Weinberg
equilibrium (Nei 1973), and is computed as
where I is the number of distinct alleles at a locus, and pi
(i = 1, 2, …, I) is the frequency of allele i in the popula-
tion. The structure of CaLas populations was determined
with dendrograms built by MEGA X software (Kumar etal.
2018) with the imported genetic distance matrix data using
H
=1−∑
I
i=1
p
2
i
the unweighted paired group method with arithmetic mean
(UPGMA) in POPGENE v 1.32.
CRISPR/cas analyses
In this study we also tried to find the presence of CRISPR/
cas system in CaLas isolates. The CRISPR repeats array in
the prophage region of different CaLas isolate were iden-
tified by PCR amplification with primer set CRIF/CRIR
(Zheng etal. 2016a) (Table1). Amplicons were directly
sequenced bi-directionally by standard Sanger’s sequenc-
ing method through a commercial DNA sequencing service
(Eurofins, Bangalore, India). Multiple sequence alignment
was performed using the BioEdit software (Hall 1999).
Results
Detection ofCaLas incitrus samples
A total of 1018 citrus trees from 18 states of India (North-
East, North-West, Central and South) were sampled cov-
ering all the predominant citrus species (Supplementary
TableS1). Presence of CaLas was confirmed by 16S rRNA
gene based qPCR method of Li etal. (2006) using primer
set HLBas/HLBr and probe HLBp. A total of 441 citrus
tissue samples out of 1018 samples were tested positive
(qPCR threshold cycle (Ct) value < 32) for CaLas. (Ct)
value ranged from 17.32–30.91 in different HLB positive
citrus samples (Supplementary TableS1). The detection of
CaLas was also confirmed by conventional PCR amplifica-
tion of an expected 500 bp DNA amplicon with primer set
Las606/LSS. No PCR products were observed in healthy
tissues (Supplementary Fig. S2). Some of the representative
cPCR amplicons were directly sequenced and the subsequent
NCBI BLAST searches confirmed the identification of the
obtained sequences to Ca. L. asiaticus (Data not shown).
These 441 CaLas positive DNA samples (isolates) were
obtained from 18 citrus growing states of India (Assam-
57, Gujarat-10, Nagaland-12, Manipur-9, Mizoram-15,
Arunachal Pradesh-11, West Bengal-23, Rajasthan-16,
Madhya Pradesh-68, Andhra Pradesh-19, Telangana-15,
Karnataka-24, Maharashtra-69, Punjab-39, Tamilnadu-14,
Meghalaya-17, Sikkim-12 and Tripura-11) and were used
in the study for screening of prophage types and assessing
CaLas population diversity where each sample typically rep-
resents a single tree.
Detection ofprophages byPCR andprevalence
ofdifferent prophage combinations
All possible eight combinations of the three known prophage
types (Type 1, Type 2 and Type 3) were detected (Table2)
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World Journal of Microbiology and Biotechnology (2021) 37:95
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95 Page 8 of 14
using different prophage -specific PCR markers. The high-
est 117 samples (26.5%) harboured only Type 1 prophage,
followed by 90 samples (20.4%) of the combination Type
1 + Type 2 prophage and then 83 samples (18.8%) showed
only Type 2 prophage. The combinations of all the 3
prophages i.e. Type 1 + Type 2 + Type 3 were noticed in 50
samples (11.3%). The number of CaLas isolate lacking all
three prophages was found to be 19 (4.3%). All the probable
eight combinations were found in the states of Karnataka,
Telangana, Maharashtra and Madhya Pradesh. The combi-
nation Type 1 + Type 3 prophage accounted for 55 samples
(12.5%) whereas Type 2 + Type 3 combination accounted
for 21 samples (4.8%). Presence of Type 3 alone accounted
lowest 6 samples (1.4%). In the Western part of India, in the
state of Gujarat, only Type 2 prophage was found (Table2).
Supplementary Fig. S2 illustrates the representative ampli-
cons obtained by using two each of Type 1, Type 2 and Type
3 prophage specific primer sets. In addition, among eight
different types of prophage combination groups of CaLas
isolates, no particular citrus cultivar preference was found,
e.g., three different types of prophage combination were
identified from 20 HLB-affected Nagpur mandarin (Citrus
reticulata) trees in the same orchard located in Maharash-
tra and Type 1 CaLas strains were confirmed from the 15
DNA samples extracted from three different citrus species
(C. reticulata, C. grandis, C. jambhiri) in the same orchard
located in Assam.
Sequencing ofprophage regions andphylogenetic
analysis
The amplified and sequenced 18 Prophage regions were
submitted to the NCBI GenBank database with the Acc.
Nos. MN650709- MN650710 and MN650712-MN650716
(Type 1), MN 650717-MN650723 (Type 2), MN660063-
MN660066 (Type3). Additional nine CaLas phage
sequences (JF773396, HQ377373, KX879602, CP010804,
CP001677, HQ377372, HQ377374, KY661963, CP019958)
were obtained from GenBank database and used for phylo-
genetic tree construction. The resulting tree depicting the
phylogenetic relationship of all 27 isolates/strains, based
on the partial nucleotide sequences alignment of differ-
ent Prophage-type regions, revealed three distinct clusters
(Fig.3), supported by high bootstrap values. The prophages
were clustered along with the line of prophage types repre-
sented by Type 1 (SC1-like), Type 2 (SC2-like) and Type 3
(P-JXGC-3) (Fig.3).
CRISPR/cas analyses
PCR-based detection method using the primer pair CRIF/
CRIR identified a CRISPR (clustered regularly inter-
spaced short palindromic repeats) region in all the 3 types
of prophages (Type 1/2/3). Thirteen CaLas field isolates
sampled from 6 states (Madhya Pradesh, Maharashtra, Kar-
nataka, Assam, Mizoram and Punjab) were found to have
a CRISPR element based on the PCR assays and subse-
quent sequence analysis. As reported earlier (Zheng etal.
2016b), the CRISPR array contained four highly similar
22 bp repeats with three heterologous spacers of 23 bp
(Fig.4). Repeat sequences were much more homogeneous
(82%, 18/22) than spacers (39%, 9/23). No similar CRISPR
array was found in GenBank sequence database except for
the already published CaLas prophages (Wang etal. 2017),
suggesting the CRISPR/cas system was shared by these
prophages.
Diversity ofCaLas populations
A pairwise population matrix of genetic distance and genetic
identity was generated for CaLas populations from individ-
ual states (Supplementary TableS2) using the POPGENE
1.32 software. By detection of Type 1, Type 2 and Type
3 prophages (Zheng etal. 2018), CaLas populations from
eighteen citrus growing states were separated into two major
Prophage Typing Groups (PTG1 and PTG2), and five sub-
groups (PTG1-A, PTG1-B, PTG2-A, PTG2-B and PTG2-C).
(Fig.5).
Fig. 3 An unrooted phylogenetic tree of “Candidatus Liberibacter
asiaticus” isolates reconstructed from three different genetic regions
(endolysin, φ structural protein gene, hsds) and based on propahge
sequences obtained by using type-specific primers, T1-2F/T1-2R,
T2-2F/T2-2R and 891-F/891-R. Downloaded sequences from pub-
lished prophages are identified in red circle, while rests are derived
from this study. Numbers at each branch are bootstrap values sup-
ported in 1000 replications by neighbour-joining method
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World Journal of Microbiology and Biotechnology (2021) 37:95
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The prophage typing group 1 (PTG1) formed from North-
West India and prophage typing group 2 (PTG2) classified
from rest of the country (North-East, Central and South
India), and both major groups were further divided into
two (PTG1-A, PTG1-B) and three (PTG2-A, PTG2-B and
PTG2-C) subgroups respectively. The subgroup PTG1-A
comprised of CaLas isolates from Punjab and Rajasthan and
PTG1-B from Gujarat, the North-West sates of India. The
PTG2-A comprised of CaLas isolates from North Eastern
states of India, although this subgroup further showed signif-
icant genetic heterogeneity within (Fig.5). The CaLas iso-
lates from Assam, Arunachal Pradesh, Meghalaya, Manipur
and Mizoram were together. Whereas, the CaLas isolates
from Nagaland, Hilly terrains of West Bengal, Sikkim and
Tripura clustered together. The subgroup PTG2-B comprised
of CaLas isolates from South and Central states of India
(Andhra Pradesh, Maharashtra, Telangana, Karnataka and
Madhya Pradesh). Interestingly, the CaLas isolates from
Tamil Nadu segregated into a distinct subcluster PTG2-C
(Fig.5).
Nei’s H-value which is based on the number of effective
alleles was used to describe the extent of heterogeneity. The
H-values of genetic diversity for CaLas populations from
Central India and adjoining Southern states were found rela-
tively higher whereas the H-values of genetic diversity for
CaLas populations from North-West and North-East Indian
states (except Arunachal Pradesh and Sikkim) were rela-
tively low. The overall average estimated H value was 0.3512
(Table3).
Discussion
One of the most fascinating characteristic discovered in
‘Ca. Liberibacter spp.’ in recent times is the presence of
prophages integrated in their genomes (Zhang etal. 2011).
Many bacterial pathogens contain prophages or phage
Fig. 4 Sequence alignment of clustered regularly interspaced short
palindromic repeats (CRISPR) arrays among 22 isolates/prophage
sequences of “Candidatus Liberibacter asiaticus”. Already published
9 prophage sequences are identified in blue. Prophage sequences
were either direct GenBank deposition (SC1, SC2, FP2, P-JXGC-3,P-
SGCA5-1) or extracted from whole genome sequences (FP1, Gen-
Bank: CP001677.5; P-A4-2, GenBank: CP010804.1; P-gxpsy-1, Gen-
Bank: CP004005.1; UF506, GenBank: HQ377374.1). MP, MS, KA,
AS, MZ and PJ represent the CaLas isolates from Madhya Pradesh,
Maharashtra, Karnataka, Assam, Mizoram and Punjab states. Strain
P-A4-2 was used as a reference. CRISPR repeats are highlighted in
yellow. Dots represent nucleotide identity to those of strain P-A4-2. A
* at the bottom of alignment indicates identical nucleotides. Nucleo-
tide variations are marked in red
Fig. 5 The grouping of ‘Candidatus Liberibacter asiaticus’ popu-
lations from eighteen citrus growing states of India by detection
of the presence of prophages. The dendrogam was built using the
unweighted paired group method with arithmetic mean (UPGMA)
and figures represent genetic distance between isolates. PTG,
prophage typing group. Abbreviations for different states are as men-
tioned in Fig.1
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World Journal of Microbiology and Biotechnology (2021) 37:95
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95 Page 10 of 14
remnants integrated in their genomes that encode virulence
factors (Menouni etal. 2014). Till date 3 distinct prophage
types were reported and described in CaLas: SC1-type or,
Type 1, SC2-type or, Type 2 and P-JXGC3-type or, Type
3. Considerable information is available on chromosomally
integrated prophage SC1 and an excision prophage SC2.
SC1 replicates and forms phage particles in the phloem of
‘CaLas’-infected periwinkle (Catharanthus roseus) and in
sweet orange plants (Fu etal. 2015). Both phages encode
two proteins with peroxidase activities (Zhang etal., 2011).
These peroxidases might protect the bacteria against reactive
oxygen species (ROS) produced by the plant during infection
(Jain etal. 2015). The SC1 prophage also encodes functional
holin (SC1_gp110) and endolysin (SC1_gp035) proteins
that might be implicated in bacterial membrane lysis and
cell-wall degradation during bacteriophage egress (Fleites
etal. 2014). Another incomplete phage/prophage variant
(iFP3) derived from recombination of FP1 (SC1) and FP2
(SC2) was reported to be associated with HLB blotchy mot-
tle symptoms and disease development (Zhou etal. 2013;
Pitino etal. 2014).
These three types of prophages, namely Type 1 (SC1,
NC_019549.1), Type 2 (SC2, NC_019550.1), and Type 3
(P-JXGC-3, KY661963.1) have been successfully used to
characterize and group CaLas strains worldwide (Puttamuk
etal. 2014; Zheng etal. 2018; Silva etal. 2019; Fu etal.
2020). The present study for the first time describes the
genetic structure of different types of prophages associated
with CaLas population infecting all major citrus cultivars
across different states in India.
A total of 441 CaLas isolates collected from 18 Indian
citrus growing states covering all major citrus pockets were
analyzed by PCR-based prophage typing and subsequently
an effort has been made to recognize a PTG-based sys-
tem for evaluation of CaLas isolates. All the three known
prophages (Type-1, -2 and -3) along with all the probable 8
prophage combinations were found prevalent in citrus pro-
duction zones of India. Prophage types were detected by
PCR and confirmed by amplicon sequencing and phyloge-
netic analyses.
With the detection of three known prophages, two core
PTGs were identified in India: first one (PTG1) from North-
West India and the second group (PTG2) classified from rest
of the country (North-East, Central and South India), and
both major groups were further divided into two (PTG1-A,
PTG1-B) and three (PTG2-A, PTG2-B and PTG2-C) sub-
groups respectively. This noteworthy difference between
PTG1 and PTG2 indicates that the two regions might have
independent origin of infections and also suggesting a pos-
sible occurrence of different introduction events in those
two regions. It is also hypothesized that evolution of CaLas
pathogen in these citrus belts might have been driven by
different and differential factors like epigenetic factors,
pathogens load and vector population. Very recently, by the
detection of three known prophages, two major PTGs were
identified in mainland China: PTG1 of HAR (high altitude
regions) and PTG2 of LAR (low altitude regions) (Fu etal.
2020). Interestingly, similar observations and suggestions
were made based on phylogenetic studies of CaLas strains
identified using different markers (Liu etal. 2011; Wang
etal. 2012; Zheng etal. 2017). Using the prophage typing
system, Dai etal. (2019) identified four prophage typing
groups (PTGs) with the California CaLas strains and sug-
gested the origin of California CaLas was Asia instead of
Florida according to a phage DNA terminate large subunit
gene (terL) gene sequence.
Though there are different opinions among research-
ers about the origin of HLB-associated CaLas, historical
evidences and early literature reviews related to HLB sug-
gested that the most ancient population of ’Ca. L. asiati-
cus’ perhaps originated from unidentified native rutaceae in
North-West part of India (Beattie etal. 2008a, b). Present
study demonstrates the predominance of PTG1 lineage of
CaLas in North-West India. It is probable that PTG1 lineage
of CaLas may have moved from North-West India to other
Asian/South-East Asian countries through infected planting
Table 3 Nei’s gene diversity (H) of “Candidatus Liberibacter asiati-
cus” populations within citrus growing states of India
Geographical origin H value
North-East India
Assam 0.2347
Nagaland 0.3796
Manipur 0.2993
Mizoram 0.3674
Arunachal Pradesh 0.4298
West Bengal (Sub-Himalayan tracts—Darjeeling and
Kalimpong Hills)
0.3453
Sikkim 0.4028
Meghalaya 0.2622
Tripura 0.3747
North-West India
Punjab 0.3068
Rajasthan 0.3125
Gujarat 0.0000
Central India
Madhya Pradesh 0.4513
Maharashtra 0.4624
South India
Andhra Pradesh 0.4875
Telangana 0.4800
Karnataka 0.4769
Tamilnadu 0.2483
Overall average 0.3512
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World Journal of Microbiology and Biotechnology (2021) 37:95
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Page 11 of 14 95
materials and Asian citrus psyllid-mediated dispersal (da
Graca 1991; 2004). PTG1 might have spread from North-
West India to other citrus pockets of India, which will be
clear once additional samples are characterized in future.
However, It is still difficult to precisely elucidate when the
disease entered each country and from where it was intro-
duced. Frequent shipment of plant materials and illegal
importation of planting materials has increased the risk of
disseminating exotic plant pathogens around the globe.
The group PTG 2-A consists of isolates predominantly
from all the North-Eastern (NE) region states. In NE states,
a geographically locked region, citrus is cultivated both in
high altitude and low altitude areas. It would be interesting
to analyse the population variation on the basis of altitudinal
differences as reported earlier by Fu etal. 2020. NE region
of India, considered as one region of origin of contempo-
rary Citrus species (Bhattacharya and Dutta 1956; Wu etal.
2018), is more likely to be a region of origin for HLB for
PTG2-A as evident from present study. Potentially, this line-
age in NE states has moved and spread to border countries,
which are geographically close to NE regions such as China,
Myanmar, Vietnam or Thailand mainly through mandarin
seedlings. However, this remains to be further confirmed as
there is a lack of both historical documents and molecular
evidence.
The wide dissemination of HLB within Central and
Southern India was partially due to propagation of trees by
grafting of scions from uncertified and inadequately indexed
mother trees, in addition to poor management of abandoned
orchards and psyllid vectors (Das etal. 2002, 2019). Some
of the popularly known nurseries situated, for example, at
Shendur-Janaghat in Maharashtra state and Railway Kodoru,
Andhra Pradesh state distribute uncertified citrus plant mate-
rial to the citrus growers which leads to rampant spread of
HLB infection across the states. Widespread transport/
movement of diseased planting material from one state to
another state also lead to distribution of CaLas genotypes.
Psyllid (D. citri) vector occurrence being more rampant in
mainland India compared to NE Hills region might be the
reason for more variability of CaLas isolates in the mainland
with respect to the prophage repertoire. However in future
it would be interesting to see whether any PTG would alter
by passage through the psyllid vector and how its dynamics
changes over the citrus growing groves.
The H values of genetic diversity for CaLas populations
from NE states were relatively low, whereas the H-values of
Central and Southern India and adjoining states were found
relatively higher. The high diversity of CaLas in Central
and Southern India could be due to higher prophage activ-
ity (as prophages are important agents of horizontal gene
transfer) and increased CaLas genome plasticity might have
been observed due to its favourable ecological conditions. It
is notable here that limited number of CaLas isolates have
been analyzed especially from North-West and NE India. In
future, analysis of additional samples from all major citrus
growing areas of India will lead to deep understanding of
CaLas prophage diversity.
The small proportion of CaLas strains without any of
the three known prophages could harbour other unknown/
uncharacterized types of prophage or no prophage at all. It
has been demonstrated that the genetic structure and acquisi-
tion of CaLas in psyllid vector is mediated by the citrus host
(Meng etal. 2018; Fu etal. 2020). Thus, the observation of
a particular prophage pattern and its frequency in different
states could be influenced by the different citrus cultivars
sampled from different regions in this study, indicating citrus
cultivar effects cannot be fully ruled out.
Our study also showed the presence of CRISPR locus in
all the 3 prophage types. Since all the 3 types were observed
in 11% of surveyed samples in our present study inferring
that 3 types of prophages coexist in citrus growing regions of
India. Considering that the function of a CRISPR/cas system
is to destroy invading DNA based on spacer information,
it can be assumed that a pre-established CaLas prophage
in a CaLas cell could use its CRISPR/cas system to defeat
the invasion of the other phage/prophage DNA. The CaLas
prophage carried an immunity structure called a CRISPR/
cas system has already been reported previously (Zheng
etal. 2016a). Along the same line, it has been also reported
that Type-3 prophage carries a CRISPR/cas system with
the spacer sequences identical or highly similar to that of
Type 1 prophages (Zheng etal. 2018), which could be a
defense mechanism against the lytic Type 1 phage/prophage.
However, experimental proofs and molecular evidences are
needed to confirm these propositions. Further, in case of
Type 3 prophages, the Restriction-Modification system was
also speculated to play a role against Type 1 prophage/phage
invasion which could enhance the pathogen survival under
field conditions (Zheng etal. 2018).
It is worth mentioning that while the results here pro-
vide insights into the origins and genetic structure of HLB-
associated CaLas, larger population analyses using other
array of molecular markers will help resolving some of the
unanswered questions on the origin and dissemination of
CaLas. With the report of new prophage types like Type 4
(Dominguez-Mirazo etal. 2019), the possibility of exist-
ence of other prophage types also could not be ruled out as
of now.
Conclusions
This study employed a prophage-region based genetic analy-
sis to characterize 441 CaLas strains obtained from 18 citrus
growing states of India. Typed by prophage specific PCR,
the CaLas population in India contained all possible eight
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World Journal of Microbiology and Biotechnology (2021) 37:95
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95 Page 12 of 14
combinations of the three prophage types, which again clas-
sified into 3 population groups or PTGs, suggesting active
prophage/phage interactions involving lytic Type 1 prophage
and lysogenic Type 2 and Type 3 prophages. To our knowl-
edge, this is the first report on evaluating the genetic vari-
ation/diversity of a large population of Ca. L. asiaticus
infected samples in India using the signature marker genes
from CaLas prophage regions. Altogether the study dem-
onstrates that the prophage typing is sensitive and effective
approach in revealing diversity of Ca. L. asiaticus isolates
which will help to generate timely information to assist
understanding the HLB epidemic and ongoing HLB man-
agement efforts in India.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s11274- 021- 03057-8.
Acknowledgements Authors are grateful to the Funding agency,
Department of Biotechnology (DBT), Govt. of India for financial
support under DBT twinning project Agri 2015/57 (Grant No. BT/
PR16723/NER/95/264/2015) and Director, ICAR- Central Citrus
Research Institute for providing the necessary laboratory facilities.
Declarations
Conflict of interest The authors declared no conflict of interest.
Ethical approval This article does not contain any studies with animals
performed by any of the authors.
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