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DETECTION, IDENTIFICATION AND CHARACTERIZATION OF

PHYTOPHTHORA SPP. INFECTING CITRUS IN INDIA

Article  in  JOURNAL OF PLANT PATHOLOGY · March 2016

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Journal of Plant Pathology (2016), 98 (1), 55-69 
Edizioni ETS Pisa, 2015 55
DETECTION, IDENTIFICATION AND CHARACTERIZATION
OF PHYTOPHTHORA spp. INFECTING CITRUS IN INDIA
A.K. Das, S. Nerkar, A. Kumar and S. Bawage
National Research Centre for Citrus, Amravati Road, 440010 Nagpur, Maharashtra, India
SUMMARY
Phytophthora species that cause root rot, foot rot, gum-
mosis and decline of citrus in India were characterized
by morphological and molecular means. P. nicotianae
was found to be the predominant species followed by P.
palmivora and P. citrophthora in the 28 nurseries and 174
orchards of five major citrus growing states of the coun-
try viz. Maharashtra, Madhya Pradesh, Punjab, Andhra
Pradesh and Sikkim. Morphological characterization of
100 Phytophthora spp. isolates revealed 12 different types
of colony pattern on V8 agar and 9 isolates of P. nicotianae
were observed as A2 mating type and rest all the 91 iso-
lates of Phytophthora spp. were of A1 mating type, while
no self-fertile isolate was observed. The species P. nicoti-
anae, P. palmivora, and P. citrophthora could all be clearly
identified by PCR amplification of the nuclear ribosomal
internal transcribed spacer (ITS) region followed by restric-
tion analysis with MspI, AluI and RsaI. Sequencing and
phylogenetic analyses of the ITS region, beta tubulin gene,
translation Elongation factor 1 alpha and the region con-
taining the mitochondrial cytochrome c oxidase subunit 2
gene fragment also confirmed the identity of these species.
The phylogenetic groups did not correlate with geographic
origin of the isolates. Additionally, isolates of P. nicotianae
and P. palmivora were identified using species-specific PCR
primers. The identification tools described in this study
accurately detected the species of Phytophthora affecting
citrus in nurseries and orchards in India.
Key words: P. nicotianae, P. palmivora, P. citrophthora,
ITS-RFLP, sequencing
INTRODUCTION
Citrus is the third largest fruit industry after banana
and mango in India, with an estimated production of
9638 thousand tonnes of fruits and area coverage of 1001
Corresponding author: A.K. Das
E-mail : dasashiskumar@hotmail.com
thousand hectares (FAO, 2013). Citrus fruits, cultivated
under varied agro-ecological conditions, from arid and
semiarid areas of southwest region to humid subtropi-
cal climate of north-eastern region of India, are presently
plagued with various problems. Among these, heavy inci-
dence of Phytophthora- induced diseases is one of the ma-
jor causative factors for decline and short life span of citrus
orchards throughout India (Naqvi, 2004; Das et al., 2011).
The genus Phytophthora (meaning plant-destroyer in
Greek) belongs to the class Oomycetes, currently placed
in the kingdom Stramenopila, under the phylum Hetero-
konta (Cavalier-Smith and Chao, 2006; Riisberg et al.,
2009) containing about 123 formally described species
and 23 provisional species (http://www.phytophthoradb.
org/ accessed April 21, 2015). Species of Phytophthora are
distributed worldwide and cause economically important
diseases in vegetables and fruit crops, ornamental plants
and forest trees (Erwin and Ribeiro, 1996; Agrios, 2005).
Citrus cultivation globally is the victim of Phytophthora
spp. causing heavy yield losses and tree decline. Several
Phytophthora spp. viz., P. boehmeriae (Sawada), P. cacto-
rum (Leb. And Cohn) Schroeter, P. capsici (Leonian), P.
cinnamomi (Rands), P. citricola (Sawada), P. citrophthora
(R.E.Smith and E.H. Smith) Leonian, P. dreschleri (Tuck-
er), P. hibernalis (Came), P. megasperma (Drechsler), P. ni-
cotianae Breda de Haan, (= parasitica Dastur), P. palmivora
(Bulter) Butler, and P. syringae (Klebahn) Klebahn, have
been reported pathogenic on citrus from different citrus
growing areas of the world (Erwin and Ribiero, 1996).
However, P. nicotianae, P. palmivora, and P. citrophthora
infection in citrus is predominant (Bowman et al., 2007).
These malevolent pathogens can infect almost all parts
of the citrus plant causing damping-off of nursery seed-
lings, root rot, foot rot and gummosis of orchard trees, leaf
blight and brown rot of fruit.
Identification of Phytophthora to species level is crucial
in developing quarantine methods, disease control and
resistance breeding. Species identification was based pri-
marily on morphological features of sporangia, antheridia,
oogonia, oospore and chlamydospores along with other
criteria like cardinal growth temperature, growth rate,
colony morphology in culture media and mating behaviour
(Newhook et al., 1978; Stamps et al., 1990). However, these
methods are not reliable to assign to species level consider-
ing the vast diversity and highly overlapping morphologi-
cal characteristics among various species of Phytophthora.
56 Phytophthora spp. on Citrus
Fig. 1. Symptoms of Phytophthora-induced diseases in Nagpur mandarin (Citrus reticulata) tree in Nagpur district of Maharashtra
state, India. Collar rot and root rot (a) gummosis (b), tree decline (c).
So rapid and trustworthy approach has been realized in
the last decade, which is evident with the progress made
especially in the use of DNA-based molecular methods for
detection, identification, and classification of Phytophthora
species (Ristaino et al., 1998; Cooke et al., 2000a; Drenth et
al., 2006; Grunwald et al., 2011; Martin et al., 2014). Pres-
ently, an amalgamation of morphological and DNA-based
methods has been employed to precisely identify the genus
Phytophthora at species level (Gallegly and Hong, 2008;
Kroon et al., 2012).
Disease management of Phytophthora is heavily reliant
on prevention of infection through the production of clean
planting material, disease-free soil, high levels of nursery
hygiene, resistant rootstock, and improving drainage and
soil health in the field. Since hygiene is a relevant part of
integrated disease management of Phytophthora, it is im-
portant to accurately determine the presence of Phytoph-
thora in plant material, potting mixes and soil samples.
The use of disease-free planting material is not only impor-
tant in halting the spread of Phytophthora pathogens from
nurseries to orchards and fields but also highly relevant to
limiting the spread of these fungal-like pathogens across
the globe. Precise detection, diagnosis and species identifi-
cation at molecular level particularly with the aid of DNA
markers and sequencing would certainly boost productiv-
ity by curbing the dreaded pathogen with appropriate con-
trol measures. However, this is possible by understanding
the distribution and monitoring of Phytophthora species
and learning their variation in natural ecosystems. In the
present study a number of Phytophthora species isolates
from various citrus cultivars were isolated from different
citrus growing places in India and characterized morpho-
logically as well as by molecular methods (PCR RFLP, am-
plification of multiple loci, sequencing and phylogenetic
analysis) to assess the diversity present therein.
Journal of Plant Pathology (2016), 98 (1), 55-69
MATERIALS AND METHODS
Survey, isolation, purification and maintenance of
pathogen isolates. One hundred Phytophthora spp. iso-
lates were obtained from 174 citrus orchards and 28 cit-
rus nurseries during the year 2008 through 2010 situated
in Vidarbha and Marathwada region (Nagpur, Amravati,
Jalna, Pune districts) of Maharashtra state, Chhindwara
district of Madhya Pradesh state, Hoshiarpur region of
Punjab state, Nalgonda district of Andhra Pradesh state
and eastern part of Sikkim states (Table 1). Soil, root, leaf,
bark and fruit samples were collected from mild to severe-
ly infected plants (showing symptoms of foot and collar
rot, root rot, gummosis and decline) of Nagpur mandarin
(Citrus reticulata) (Fig. 1), Kinnow mandarin (C. reticulata),
Sikkim mandarin (C. reticulata), acid lime (C. aurantifolia),
Mosambi / Sathgudi sweet orange (C. sinensis), pummelo
(C. grandis) and trifoliate orange (Poncirus trifoliata). Nag-
pur mandarin and Mosambi sweet orange cultivars are
generally grown on rough lemon (C. jambhiri) or Rang-
pur lime (C. limonia) rootstocks. From the ground bed
and containerized nurseries in the field, damping off,
collar and root rot infected citrus samples were collected
for pathogen isolation. In the orchard, soil samples were
collected from the rhizosphere of each plant at 15-30 cm
depth along with feeder roots.
For isolation of Phytophthora from roots, samples were
washed with sterile distilled water, and dried up with
sterilized filter paper. Fibrous roots (< 2 mm in diameter)
were cut in 5 mm pieces and inserted into the surface of
Corn meal agar (CMA, HiMedia Laboratories, Mumbai,
India) pimaracin-ampicilin-rifampicin-PCNB-hymexazol
(PARPH) selective medium (Kannwischer and Mitchell,
1978) plates. For isolation of Phytophthora from gummosis
lesions, affected bark tissue samples were washed under
Journal of Plant Pathology (2016), 98 (1), 55-69 Das et al. 57

Table 1. Collection data and summary information about the Phytophthora spp. isolates examined in this study.

Colony
Place of collection Month/Year Mating
Isolate Code No. Phytophthora spp. Sample typea Hostb Morphology
Locality/District/State of isolation type
typec
NRCPh-1 P. nicotianae Patansawngi/ Nagpur/ Maharashtra 06/2008 Soil NM A1 IIE
NRCPh-2 P. nicotianae NRCCd/ Nagpur/ Maharashtra 06/2008 Soil NM A1 IIA
NRCPh-3 P. nicotianae Katol/ Nagpur/ Maharashtra 06/2008 Fruit MO A1 IIA
NRCPh-4 P. nicotianae NRCC/ Nagpur/ Maharashtra 06/2008 Soil NM A1 IID
NRCPh-5 P. nicotianae Chandur bazaar/ Amaravati/ Maharashtra 08/2008 Soil NM A1 IIA
NRCPh-6 P. nicotianae Bhibhapur/ Nagpur/ Maharashtra 08/2008 Root NM A1 IIB
NRCPh-7 P. nicotianae Jalalkheda/ Nagpur/ Maharashtra 08/2008 Soil MO A1 VB
NRCPh-8 P. nicotianae Achalpur/ Amaravti/ Maharashtra 09/2008 Fruit NM A1 VA
NRCPh-9 P. nicotianae NRCC/ Nagpur/ Maharashtra 09/2008 Soil NM A1 IIB
NRCPh-10 P. nicotianae Gondkhairi/ Nagpur/ Maharashtra 10/2008 Soil NM A1 IIB
NRCPh-11 P. nicotianae Mohpa/ Nagpur/ Maharashtra 10/2008 Fruit NM A1 IIB
NRCPh-12 P. nicotianae Mansar/ Nagpur/ Maharashtra 10/2008 Soil NM A1 IIA
NRCPh-13 P. palmivora NRCC/ Nagpur/ Maharashtra 10/2008 Soil NM A1 ID
NRCPh-14 P. palmivora Narkhed/ Nagpur/ Maharashtra 10/2008 Soil NM A1 ID
NRCPh-15 P. palmivora Amravati road Nursery/ Nagpur/ Maharashtra 05/2009 Soil RO A1 ID
NRCPh-16 P. nicotianae Amravati road Nursery/ Nagpur/ Maharashtra 05/2009 Soil RA A1 IIA
NRCPh-17 P. nicotianae Amravati road Nursery/ Nagpur/ Maharashtra 05/2009 Soil NM A1 IIE
NRCPh-18 P. nicotianae Mohpa/ Nagpur/ Maharashtra 06/2009 Soil NM A2 IIA
NRCPh-19 P. nicotianae NRCC/ Nagpur/ Maharashtra 05/ 2009 Soil NM A2 IIB
NRCPh-20 P. palmivora NRCC Nagpur/ Maharashtra 05/ 2009 Soil NM A1 IA
NRCPh-21 P. palmivora NRCC/ Nagpur/ Maharashtra 05/ 2009 Soil NM A1 IA
NRCPh-22 P. nicotianae NRCC/ Nagpur/ Maharashtra 05/ 2009 Soil NM A1 IID
NRCPh-23 P. palmivora NRCC/ Nagpur/ Maharashtra 06/ 2009 Soil NM A1 IA
NRCPh-24 P. nicotianae NRCC/ Nagpur/ Maharashtra 06/ 2009 Soil NM A2 IID
NRCPh-25 P. palmivora NRCC/ Nagpur/ Maharashtra 06/ 2009 Soil NM A1 IA
NRCPh-26 P. palmivora NRCC/ Nagpur/Maharashtra 06/ 2009 Soil NM A1 ID
NRCPh-27 P. palmivora NRCC/ Nagpur/ Maharashtra 08/ 2009 Soil AL A1 IA
NRCPh-28 P. palmivora NRCC/ Nagpur/ Maharashtra 08/ 2009 Soil PU A1 IA
NRCPh-29 P. palmivora NRCC/ Nagpur/ Maharashtra 08/ 2009 Soil PU A1 IA
NRCPh-30 P. palmivora Amravati road Nursery/ Nagpur/ Maharashtra 08/ 2009 Soil RO A1 IA
NRCPh-31 P. nicotianae NRCC glasshouse/ Nagpur/ Maharashtra 08/ 2009 Soil RO A1 IIA
NRCPh-32 P. nicotianae Fetri/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 IIA
NRCPh-33 P. nicotianae Fetri/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 IIA
NRCPh-34 P. nicotianae Brahmni/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 IIA
NRCPh-35 P. nicotianae Pimpla Kinkhede/ Nagpur/ Maharashtra 08/ 2009 Soil NM A2 IIA
NRCPh-36 P. nicotianae Pimpla Kinkhede/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 IIA
NRCPh-37 P. nicotianae Pimpla Kinkhede/ Nagpur/ Maharashtra 08/ 2009 Soil NM A2 IIA
NRCPh-38 P. nicotianae Pimpla Kinkhede/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 VA
NRCPh-39 P. nicotianae Pimpla Kinkhede/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 VA
NRCPh-40 P. palmivora Pimpla Kinkhede/ Nagpur/ Maharashtra 09/ 2009 Fruit NM A1 IA
NRCPh-41 P. nicotianae Hatla/ Nagpur/ Maharashtra 08/ 2009 Soil AL A1 IIA
NRCPh-42 P. nicotianae Chargaon/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 VB
NRCPh-43 P. nicotianae Ghorad/ Nagpur/ Maharashtra 08/ 2009 Soil NM A2 IIA
NRCPh-44 P. nicotianae Fetri/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 IIA
NRCPh-45 P. nicotianae Brahmni Nursery/ Nagpur/ Maharashtra 08/ 2009 Soil NM A2 IIA
NRCPh-46 P. nicotianae Pimpla Kinkhede/ Nagpur/ Maharashtra 08/ 2009 Soil NM A1 VB
NRCPh-47 P. nicotianae Pimpla Kinkhede/ Nagpur/ Maharashtra 08/ 2009 Soil RO A1 VA
NRCPh-48 P. nicotianae Amravati road Nursery/ Nagpur/ Maharashtra 08/ 2009 Root NM A1 IIB
NRCPh-49 P. nicotianae Dhamangaon/ Amravati/ Maharashtra 08/ 2009 Soil NM A1 IIB
NRCPh-50 P. nicotianae Dhamangaon/ Amravati/ Maharashtra 08/ 2009 Soil NM A1 IIA
NRCPh-51 P. nicotianae Jambazar, Pusad/ Yavatmal/ Maharashtra 09/ 2009 Soil NM A1 IIA
NRCPh-52 P. nicotianae Jambazar, Pusad/ Yavatmal/ Maharashtra 09/ 2009 Soil NM A1 IIA
NRCPh-53 P. nicotianae Morgaon/ Amravati/ Maharashtra 09/ 2009 Soil RO A1 IID
NRCPh-54 P. nicotianae Valki/ Amravati/ Maharashtra 09/ 2009 Soil RO A1 VB
NRCPh-55 P. nicotianae Pimpla Kinkhede/ Nagpur/ Maharashtra 09/ 2009 Leaf NM A1 VB
NRCPh-56 P. nicotianae Redni, Baramati/ Pune/ Maharashtra 09/ 2009 Soil MO A1 IIA
NRCPh-57 P. nicotianae Hingna/ Nagpur/ Maharashtra 10/ 2009 Root NM A1 IIA
NRCPh-58 P. nicotianae Mekehainwale/ Hoshiarpur/ Punjab 10/ 2009 Soil KM A1 IIA
NRCPh-59 P. nicotianae Mekehainwale/ Hoshiarpur/ Punjab 10/ 2009 Soil KM A1 IIA
NRCPh-60 P. nicotianae Badla Nursery/ Hoshiarpur/ Punjab 10/ 2009 Soil KM A2 IIB
NRCPh-61 P. nicotianae Badla/ Hoshiarpur/ Punjab 10/ 2009 Soil KM A1 IIF
NRCPh-62 P. nicotianae Chhanni Kalan/ Hoshiarpur/ Punjab 10/ 2009 Soil KM A2 IIA
NRCPh-63 P. nicotianae Badla/ Hoshiarpur/ Punjab 10/ 2009 Soil KM A1 IIA
58 Phytophthora spp. on Citrus Journal of Plant Pathology (2016), 98 (1), 55-69

NRCPh-64 P. nicotianae Loni / Amravati/ Maharashtra 11/ 2009 Root NM A1 IIF

NRCPh-65 P. nicotianae Loni / Amravati/ Maharashtra 11/ 2009 Soil NM A1 IIF
NRCPh-66 P. nicotianae Loni / Amravati/ Maharashtra 11/ 2009 Root NM A1 IIF
NRCPh-67 P. nicotianae Loni / Amravati/ Maharashtra 11/ 2009 Soil NM A1 IIF
NRCPh-68 P. nicotianae Isapur/ Amravati/ Maharashtra 11/ 2009 Root NM A1 IIA
NRCPh-69 P. nicotianae Momdapur/ Amravati/ Maharashtra 11/ 2009 Soil RO A1 IIA
Mordongre, Pandhurna/ Chhindwada/ Madhya
NRCPh-70 P. nicotianae 11/ 2009 Root NM A1 IIA
Pradesh
Mordongre, Pandhurna/ Chhindwada/ Madhya
NRCPh-71 P. nicotianae 11/ 2009 Root NM A1 IIA
Pradesh
NRCPh-72 P. nicotianae Kuddam/ Chhindwada/ Madhya Pradesh 11/ 2009 Root NM A1 IIA
NRCPh-73 P. nicotianae Berdi / Chhindwada/ Madhya Pradesh 11/ 2009 Root NM A1 IID
NRCPh-74 P. nicotianae Rampeth / Chhindwada/ Madhya Pradesh 11/ 2009 Soil NM A1 IID
NRCPh-75 P. nicotianae Bhivkundi / Amravati/ Maharashtra 01/ 2010 Root NM A1 IID
NRCPh-76 P. nicotianae Mayawadi / Amravati/ Maharashtra 01/ 2010 Root NM A1 IIA
NRCPh-77 P. nicotianae Dagha / Amravati/ Maharashtra 01/ 2010 Soil NM A1 IIF
NRCPh-78 P. nicotianae Jalna/ Jalna/ Maharashtra 01/ 2010 Soil MO A1 IIF
NRCPh-79 P. palmivora NRCC/ Nagpur/ Maharashtra 12/ 2009 Roots NM A1 IA
NRCPh-80 P. nicotianae Daringa/ Hoshiarpur/ Punjab 01/ 2010 Soil KM A1 IIF
NRCPh-81 P. nicotianae Mehlanwali/ Hoshiarpur/ Punjab 02/ 2010 Soil KM A1 IIA
NRCPh-82 P. nicotianae Mehlanwali/ Hoshiarpur/ Punjab 02/ 2010 Soil KM A1 IID
NRCPh-83 P. nicotianae Mehlanwali/ Hoshiarpur/ Punjab 02/ 2010 Soil KM A1 IIA
NRCPh-84 P. nicotianae Mehlanwali/ Hoshiarpur/ Punjab 02/ 2010 Soil KM A1 IIA
NRCPh-85 P. nicotianae Mehlanwali/ Hoshiarpur/ Punjab 02/ 2010 Soil KM A1 IIA
Gummosis
NRCPh-86 P. nicotianae Amravati road Nursery/ Nagpur/ Maharashtra 03/ 2010 NM A1 ID
lesion
NRCPh-87 P. nicotianae Panchgaon, Umred/ Nagpur/ Maharashtra 07/ 2010 Leaf MO A1 IID
NRCPh-88 P. palmivora Paratwada/ Amravati/ Maharashtra 08/ 2010 Fruit NM A1 IA
NRCPh-89 P. nicotianae Nalgonda/ Nalgonda/ Andhra Pradesh 09/ 2010 Root SO A1 IID
NRCPh-90 P. nicotianae Nalgonda/ Nalgonda/ Andhra Pradesh 09/ 2010 Soil SO A1 VB
NRCPh-91 P. nicotianae Amravati road Nursery/ Nagpur/ Maharashtra 11/ 2010 Leaf NM A1 VB
NRCPh-92 P. palmivora NRCC/ Nagpur/ Maharashtra 12/ 2010 Soil RA A1 IA
NRCPh-93 P. palmivora NRCC/ Nagpur/ Maharashtra 12/ 2010 Root TO A1 IA
NRCPh-94 P. palmivora NRCC/ Nagpur/ Maharashtra 12/ 2010 Soil PU A1 ID
NRCPh-95 P. nicotianae NRCC/ Nagpur/ Maharashtra 12/ 2010 Root HR A1 VB
NRCPh-96 P. nicotianae Mohgaon/ Chindwada/ Madhya Pradesh 12/ 2010 Soil NM A1 IIA
NRCPh-97 P. citrophthora Sakhu Nursery/ East district/ Sikkim 12/ 2010 Soil SM A1 IV
NRCPh-98 P. nicotianae Mazithar Nursery/ East Sikkim / Sikkim 12/ 2010 Soil SM A1 IIA
NRCPh-99 P. nicotianae Mazithar Nursery/ East Sikkim / Sikkim 12/ 2010 Soil SM A1 VB
NRCPh-100 P. palmivora NRCC/ Nagpur/ Maharashtra 12/ 2010 Soil RA A1 VC
a 
Soil = Rhizosphere soil.
b 
NM-Nagpur mandarin; SM-Sikkim Mandarian; RO-Rough Lemon, RA-Rangpur Lime, MO-Mosambi (Sweet Orange), SO-Sathgudi Orange, KM-
Kinnow Mandarian, AL-Acid Lime, PU-Pummello, TO-Trifoliate Orange, HR-(Rough lemon X Trifoliate) Hybrid Rootstock.
c  Colony morphology pattern as observed on V8 agar (modified after Appiah et al. 2003). IA, stellate striated pattern; ID, stellate pattern; IIA, cottony
mycelium, no pattern; IIB, dense cottony mycelium, no pattern; IID, cottony with slightly petalloid pattern; IIE, cottony mycelium with concentric ring
growth pattern; IIF, fluffy cottony mycelium, no pattern; III, cottony mycelium with slightly stellate pattern; IV, Petalloid pattern; VA, dense rosette pat-
tern and VB, light rosette pattern; VC, Rosaceous pattern.
d  National Research Centre for Citrus (NRCC) farm site.

running tap water, surface sterilized by 5-10 seconds im-
mersion in 70% ethanol and dried on filter paper. Af-
fected tissues from the edges of lesions were selected by
cutting 2-4 mm wide pieces which were placed on modi-
fied PARBPH selective agar (Alvarez et al., 2008) contain-
ing CMA; this same procedure was followed for isolation
of Phytophthora from infected fruits. For isolation of the
pathogen from soil, 10 cm3 of soil sample was suspended
in 90 ml water supplemented with 0.25% of agar powder.
One ml of this suspension was spread on PARPH medi-
um and plates were incubated for 48 hours at 25°C. Plates
were washed with distilled water to remove the residual
soil (Timmer et al., 1988) to observe the Phytophthora
colonies. Agar discs 6 mm in diameter from peripheral
mycelia of Phytophthora were then placed on fresh PARPH
medium for subsequent purification. Baiting (Erwin and
Ribeiro, 1996) with rough lemon (C. jambhiri) leaf bits was
followed to isolate Phytophthora spp. from the soil samples
which failed to produce any colony in direct plating meth-
od. Cultures were then subcultured until pure cultures
were obtained. Purified colonies from PARPH were then
transferred to CMA. All the isolates were routinely main-
tained on CMA medium at 25°C with bimonthly (once
every two months) transfer to fresh medium. A culture of
each isolate was stored in sterile distilled water at room
temperature for long-term preservation.
Morphological and phenotypic characterization. Spo-
rangium morphology was checked through agar-disc-in-
water technique (Al-Hedaithy and Tsao, 1979). Briefly, agar
Journal of Plant Pathology (2016), 98 (1), 55-69 Das et al. 59

Table 2. Details of oligonucleotide primers used in this study.

Annealing conditions
Locus Primer Sequence (5’- 3’) Temperature Time (s) Reference
(°C)
ITS4 TCC TCC GCT TAT TGA TAT GC White et al. 1990
rDNA ITS region 54 30
ITS6 GAA GGT GAA GTC GTA ACA AGG Cooke et al. 2000a
FM35 CAG AAC CTT GGC AAT TAG G Martin 2000
COX 2 gene 48 30
FMPhy (10b) GCA AAA GCA CTA AAA ATT AAA TAT AA Martin et al. 2004
BTUBF2 CGG TAA CAA CTG GGC CAA GG
β-tubulin BTUBR2 GAT CCA CTC AAC GAA GTA CG
60 30 Kroon et al. 2004

EF1F TCA CGA TCG ACA TTG CCC TG

Translation elongation factor 1-α 62 30 Kroon et al. 2004
EF1R ACG GCT CGA GGA TGA CCA TG
PNIC 1 CAA TAG TTG GGG GTC TTA TT
rDNA ITS region 60 30 Grote et al. 2002
PNIC 2 GTA TAC CGA AGT ACA CAT TAA G
Pal1s CAC GTG AAC CGT ATC AAA ACT
rDNA ITS region 65 30 Tsai et al. 2006
Pal2a CAA TCA TAC CAC CAC AGC TGA

plugs (6 mm diameter) cut from the advancing margin of
colonies in CMA medium were placed in petri dishes con-
taining 20 ml sterile distilled water, and incubated for 2-3
days at 25°C under continuous white light. These plugs
were observed under the microscope to investigate sporan-
gial morphology with respect to number of papilla, shape,
caducity, type of branching, length (L), breath (B) and L/B
ratio, and chlamydospore production, shape and size.
Colony morphology was recorded as pattern and
growth rate (average daily increase in colony diameter
over the 4-day period expressed in mm day –1) of isolates
(Appiah et al., 2003; Brasier and Griffin, 1979) on V8 juice
agar (HiMedia Laboratories, Mumbai, India) and Potato
Dextrose Agar (PDA, HiMedia Laboratories, Mumbai,
India) plates (9 cm diameter petri dishes) after 4 days of
growth at 25°C in the dark. Each isolate was assessed in
triplicate.
Determination of mating type. Mating or compatibility
type of isolates was determined by the single unknown
isolate method (Kaosiri et al., 1980). A plug of 6 mm di-
ameter from each isolate was paired with a known A1 and
A2 tester of P. nicotianae (ATCC MYA-4036 and ATCC
MYA-4037 respectively, obtained from American Type
Culture collection, Manassa, VA, USA) on carrot agar
(200 g of blended carrots, 15 g of agar in 1000 ml H2O)
plates 20-25 mm apart from each other. The plates were
sealed and incubated at 20°C for 3-7 days in dark. Slides
were prepared from the line produced at the junction of
the two colonies and examined by light microscopy. If oo-
spores are seen on the plate with the known A1, but not
on the known A2 mating type, the unknown isolate is A2.
If the opposite is seen, the unknown isolate is A1 mating
type. Isolates producing oospores with both known A1
and A2 were designated A1A2. These could be self-fertile
isolates, which were confirmed by single sporangia iso-
lation to prevent from mixing cultures. Antheridial size
(L × B), oogonial average diameter (µm), oospore average
diameter (µm), number of oogonia per microscopic field
(40×) were measured for each isolate.
DNA extraction. For DNA extraction, 6 mm agar
discs of actively growing mycelium from single hyphal tip
cultures of Phytophthora spp. isolates were transferred to
25 ml of V8 broth in a conical flask and incubated in dark
for 3-4 days at 25°C. Mycelial mats were washed with ster-
ile distilled water, dried briefly under vacuum and were
ground in liquid nitrogen into fine powder with a sterile
mortar and pestle. Total genomic DNA from ca. 100 mg of
ground mycelium was extracted using DNeasy Plant mini
kit (Qiagen Inc, Valencia, CA) according to manufacturer’s
directions. DNA concentrations were estimated using a
Nanovue plus spectrophotometer (GE Life Sciences, USA).
Primers and PCR amplification. Several primer pairs
were employed for PCR amplification (Table 2). The ITS
regions of the isolates were amplified using the univer-
sal primers ITS-6 and ITS-4. Fragments of the EF-1α and
the β-tub genes were amplified using the primers EF1F/
EF1R and BTUBF2/ BTUBR2, respectively. The region
containing the mitochondrial cytochrome c oxidase sub-
unit 2 (cox2) gene fragment was amplified using FM35
and FMPhy(10b) primers. The species-specific primer
pairs PNIC 1 / PNIC 2 and Pal1s ⁄ Pal2a (Table 2) were
also used for amplifications of the ITS region (ITS1-5.8S
rRNA-ITS2) specific for P. nicotianae and P. palmivora, re-
spectively. All primers were purchased from Imperial Life
Sciences, Chandigarh, India (supplier of IDT, USA oligos).
Each PCR reaction contained 1× PCR buffer, 2 mM
MgCl2, 200 μM each dNTP, 0.5 μM of each primer, 1.25 U
of Red Taq DNA polymerase (Bioline USA Inc, Taunton,
MA), and 1 μl of template DNA. The PCR reaction mix
was adjusted to a final volume of 25 μl with nuclease-free
water (Fermentas, MD, USA). PCR amplifications were
performed on a 2720 Thermal Cycler (Applied Biosys-
tems, USA) using the following cycling protocols: an initial
60 Phytophthora spp. on Citrus Journal of Plant Pathology (2016), 98 (1), 55-69

Table 3. Phytophthora species sequence accession numbers submitted to GenBank in this study.

GenBank accession numbers

Phytophthora spp. Isolate
ITS β-tubulin EF-1α COX2
P. nicotianae NRCPh-1 HM807369
NRCPh-2 JF792525
NRCPh-3 JF792526
NRCPh-4 HM807370
NRCPh-5 JF792527
NRCPh-6 HM807371
NRCPh-7 HM807372
NRCPh-9 JF792528
NRCPh-16 JF792529
NRCPh-17 JF792530
NRCPh-18 JF792531* JN203066* JN257115*
NRCPh-19 JF792532
NRCPh-22 JF792533
NRCPh-24 JF792534
NRCPh-31 JF792535
NRCPh-37 JF792536
NRCPh-41 JF792537
NRCPh-52 JF792538
NRCPh-56 JF792539 JN203068 JN257116
NRCPh-58 JF792540 JN203069 JN257117
NRCPh-61 JF792541 KF384190 KF515637
NRCPh-66 JF792542 JN203070 JN257118
NRCPh-70 JX965375* KC984194* KC984198* JN383428*
NRCPh-71 JX965376 JN203071 JN257119
NRCPh-76 JX965377 KF384191 KF515638 JN383429*
NRCPh-81 JX965379* KC984195* KC984199* JN383431*
NRCPh-89 JN559843* KC984196* KC984200*
NRCPh-98 JN559846 KC984197 KC984201
NRCPh-99 JX965380* JN807439* KC984202* JN865174*
P. citrophthora NRCPh-97 JN559845* JN807438* JN807442* JN865173*
P. palmivora NRCPh-15 JF792543* JN203065* JN257114*
NRCPh-20 JF792544
NRCPh-21 JF792545
NRCPh-23 JF792546
NRCPh-27 JF792547* JN203067*
NRCPh-29 JF792548
NRCPh-79 JX965378* JN383430*
NRCPh- 94 JN559844
NRCPh-100 KF010299* JN807440* JN807443* JN865175*
* sequences used in phylogenetic studies.

denaturing step of 94°C for 3 min; 35 cycles of 94°C for
30 s, annealing at different conditions for various primers
used in this study as mentioned in Table 2, an extension
at 72°C for 1 min; and final extension of 72°C for 10 min.
The PCR products were visualized in 1.5% agarose gels
and documented.
ITS-RFLP. A 10 µl sample of the ITS PCR products
was separately digested with the restriction endonucleases
MspI, AluI and RsaI (Fermentas, MD, USA), according to
the enzyme manufacturer’s instructions and the restricted
products were electrophoresed in 2.5% agarose gel (Am-
resco LLC, Ohio, USA) in 1× Tris-acetate-EDTA (TAE)
buffer (containing 0.1 μg of ethidium bromide ml−1) for
60-90 min at 10 V cm−1 and visualized under UV light and
the images were recorded by a gel documentation system
(Biovis, Mumbai, India).
Sequencing and phylogenetic analysis. PCR products
(ITS, EF-1α, β-tub and cox2) were purified with QiaQuick
PCR purification kit (Qiagen), according to the manufac-
turer’s protocol and sequenced in both directions at the
commercially available automated DNA sequencing fa-
cility (Chromous Biotech Pvt Ltd, Bengaluru, India). Se-
quence trace data from all four regions mentioned above
were assigned base calls using Phred/ FinchTV software,
trimmed and assembled. 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 prior to phylogenetic
analyses to identify the closest related sequences.
For phylogenetic inference, 24 rDNA of ITS region
sequences (10 from this study and 14 from NCBI Gen-
Bank database), 20 β-tub gene sequences (9 from this study
and 11 from NCBI GenBank database), 16 Ef-1α gene
Journal of Plant Pathology (2016), 98 (1), 55-69 Das et al. 61

Table 4. Phytophthora species sequence accession numbers obtained from GenBank database used in the phylogenetic analyses.

Phytophthora spp. Gene Locus

ITS β-tubulin EF-1α COX2
GenBank GenBank GenBank GenBank
Isolate Isolate Isolate Isolate
Acc. No. Acc. No. Acc. No. Acc. No.
WPC:6711D1330 GU259318 P582 AY564081 P6303 EU080600 P1577 JF707034
P. nicotianae WPC:1443D1523 GU259177 P6303 EU080599 OV3W KF290389 Anthurium JF707018
TARI 24277 GU111670 OVF KF290379 STE-U6273 GU191189 P7665 JF707019
CH98Y1A AB367355 P16823 JF707148 P0255 EU080340 P7387 JF707021
SA3_1 DQ182739 P1577 JF707130
C301 JF707122
CH98U6B AB366376 STE-U7432 JN605858 CBS 274.33 AY564112 P6310 GU318300
K3 AM237441 NJB2013-HR-27 KJ631576 P631 EU080539 P10368 JF707021
P. citrophthora pa114 AY228577 PCRh7-1 HM752783
91-3 DQ335629
M189 GU993889
TARI 24270 GU111664 P0255 GU222099
P. palmivora P0255 EU080339 K4r HQ659674 P0113 GU222098
P44b GU993909
P0739 JF771544
Pythium sp. PI-5 DQ364465 Py37 DQ071293 CHE-287 EU199087 C5-1-1 AB108017

sequences (8 from this study and 8 from NCBI GenBank
database) and 17 cox2 gene sequences (7 from this study
and 10 from NCBI GenBank database) of Phytophthora
spp. isolates (Table 3, Table 4) were evaluated using the
software MEGA version 5.01 (Tamura et al., 2011). Dis-
tance matrices between all the pairs of sequence (of all
four regions) from the multiple alignments were calculated
and generation of phylogenetic tree was done by neighbour
joining (NJ) method (Saitou and Nei, 1987). Evolution-
ary distances were calculated using Kimura2- parameter
method (Kimura, 1980). The NJ tree and statistical con-
fidence for each tree was evaluated by bootstrap analysis
(1000 pseudoreplicates) using MEGA version 5.01 (Tamura
et al., 2011). Nucleotide sequence of Pythium sp. (for all the
4 loci) was treated as an outgroup.
RESULTS
Collection and maintenance of isolates. Out of 296
samples collected from 174 citrus orchards and 28 nurser-
ies, a total of 100 Phytophthora spp. isolates (designated
as NRCPh 1 – NRCPh 100) were isolated from five states
of India during 2008-2010 (Table 1). A total of 80 P. nico-
tianae, 19 P. palmivora and 1 P. citrophthora isolates were
purified and maintained in sterile distilled water and on
CMA plates.
Morphological characterization. Colony characteristics.
Twelve types of colony pattern were observed on V8 agar -
stellate striated pattern (IA), stellate pattern (ID), cottony
mycelium with no pattern (IIA), dense cottony mycelium
with no pattern (IIB), cottony with slightly petalloid pat-
tern (IID), cottony mycelium with concentric ring growth
pattern (IIE), fluffy cottony mycelium with no pattern
(IIF), cottony mycelium with slightly stellate pattern (III),
petalloid pattern (IV), dense rosette pattern (VA), light
rosette pattern (VB) and rosaceous pattern (VC) (Table
1). Representative colony morphologies of P. nicotianae, P.
palmivora and P. citrophthora isolates are shown in Fig. 2.
Out of 80 P. nicotianae isolates, 39 isolates had pattern IIA,
8 isolates had pattern IIB, IID pattern was shared by 10
isolates, IIE pattern was common in 2 isolates, 8 isolates
had IIF pattern, VA pattern was observed in 4 isolates, 9
isolates had pattern VB. Of 19 P. palmivora isolates, 13
isolates had IA, 5 isolates found to have ID and one isolate
had VC pattern. The only P. citrophthora isolate showed
IV type pattern.
However, on PDA (data not shown), the colonies of P.
nicotianae isolates showed dense cottony mycelium to cot-
tony mycelium to slightly stellate pattern growth whereas
P. palmivora isolates produced a stellate striated pattern
colonies. More variability was observed in the colony mor-
phology of P. nicotianae isolates than in P. palmivora on
V8 agar. In P. nicotianae isolates, total 7 different types of
colony patterns on V8 agar medium and 7 different types
of colony patterns on PDA were observed whereas in P.
palmivora isolates 3 different colony pattern on V8 agar
medium and 5 different colony patterns on PDA (data not
shown) were observed. The average growth rate (deter-
mined at 25°C) on V8 and PDA varied from 3.57-17.12 mm
day−1 for all Phytophthora spp. isolates (Table 5).
Sporangial morphology. P. nicotianae isolates produced
spheroid sporangium that were noncaducous and papil-
late (Fig. 3a), whereas P. palmivora isolates produced ovoid
papillate sporangia (Fig. 3c), mostly on sympodial sporan-
giophores, that were caducous and with short pedicel.
The sporangial characteristics with other morphological
parameters for the Phytophthora isolates are described in
Table 5. Ovoid, spherical/globose, limoniform, obturbi-
nate, ellipsoid were the most common sporangial shapes
62 Phytophthora spp. on Citrus Journal of Plant Pathology (2016), 98 (1), 55-69

Fig. 2. Colony morphology of Phytophthora spp. isolates on V8 agar. Morphology of P. nicotianae: Dense cottony mycelium, no
pattern (a) cottony with slightly petaloid pattern (b) light rossette pattern (c). Morphology of P. palmivora: stellate pattern (d) stel-
late stiated pattern (e). Morphology of P. citrophthora: pettaloid pattern (f).

Fig. 3. Sporangial and oogonial characters of Phytophthora spp isolates. Spheroid sporangium of P. nicotianae (a). Zoospore releas-
ing from sporangium (b). Ovoid sporangium of P. palmivora (c). Bipapillate sporangium of P. citrophthora (d). Smooth globose
oogonium of P. nicotianae (e), P. palmivora (f) and P. citrophthora (g).
Journal of Plant Pathology (2016), 98 (1), 55-69 Das et al. 63

observed in all Phytophthora spp. isolates investigated, in
addition P. citrophthora with bipapillate sporangium was
also observed (Fig. 3d). Chlamydospores were spherical to
ovoid, nonpapillate, thick-walled, and ranged from 24.2 to
38.8 µm in diameter (Table 5).
Determination of mating types. The mating types of
the isolates were detected in crosses with the known mat-
ing type (P. nicotianae). Among the 100 isolates, 71 P. ni-
cotianae isolates were found A1 mating type and 9 isolates
(NRCPh- 18, 19, 24, 35, 37, 43, 45, 60, 62) were observed as
A2 mating type (Table 1). All 19 P. palmivora isolates were
found as A1 mating type. The only P. citrophthora isolate
(NRCPh-97) was determined to be A1 mating type. In P.
nicotianae, P. palmivora and P. citrophthora isolates oogo-
nium was observed with an amphigynous antheridium
(Fig. 3e, 3f, 3g). The oospore formed was round and 18.8-
27.9 µm in diameter (Table 5).
PCR amplification and restriction of the ITS region
(PCR- RFLP). Polymerase chain reaction (PCR) ampli-
fication of the ITS region of the nuclear rDNA was per-
formed using the universal primers ITS4 and ITS6. The
PCR amplicons obtained from 18 representative Phytoph-
thora spp. (10 P. nicotianae, 7 P. palmivora and 1 P. citroph-
thora) isolates were about 900 bp, which was in the range
of band sizes typically observed for Phytophthora (Fig.
4a). The results of RFLP analysis of the PCR products of
all the isolates under study revealed different restriction
patterns. In case of MspI, the banding pattern of 404 bp,
390 bp (seen as one broad band of 400 bp), 120 bp in P.
nicotianae isolates, while two bands of 508 bp and 389 bp
in P. palmivora isolates and in case of P. citrophthora iso-
late (NRCPh-97), 3 bands of 371, 295 and 226 bp were ob-
served (Fig. 4b).
Correspondingly, digestion with AluI led to banding
pattern of 745 bp, 117 bp, and 52 bp (very faint) in P. nico-
tianae isolates, whereas 501 bp, 160 bp, 157 bp (latter two
bands appearing as one broad band), 42 bp (very faint) in
P. palmivora isolates and 541, 176, 175 bp restriction frag-
ments appeared in the only P. citrophthora isolate (Fig. 4c).
Similarly restriction fragment pattern obtained after diges-
tion with RsaI revealed profiles of 456 bp, 310 bp, 116 bp
and 92 bp in P. nicotianae isolates, 436 bp, 369 bp, 116 bp
in P. palmivora isolates and 426 bp, 378 bp, and 122 bp in
the P. citrophthora isolate (Fig. 4d). Our results were in
accordance with the comparative data of RFLP analysis
published earlier (Cooke et al., 2000b; Ristaino et al., 1998)
confirming the species identity in Table 1.
Detection of Phytophthora nicotianae and P. palmi-
vora using species-specific primer pairs. To validate the
identity of species, based on the PCR-RFLP results we
applied another approach by amplifying our isolates with
P. nicotianae-specific primer pair PNIC1/ PNIC2 and P.
palmivora-specific primer pair Pal1s ⁄ Pal2a. PCR ampli-
fication of genomic DNAs using the primer pair PNIC1/
PNIC 2 generated a ca. 750 bp sequence for isolate nos.
NRCPh 18, 52, 60 and 70, confirming that the pathogens
were P. nicotianae (Fig. 5a). In case of P. palmivora isolates

Table 5. Morphological characteristics of Phytophthora spp. isolates*

Parameters P. nicotianae P. palmivora P. citrophthora

No. of isolates investigated 80 isolates 19 isolates 1 isolate
Sporangia
L × B mean 41.9 ± 5.3 × 32.1 ± 4.9 44.8 ± 5.9 × 32.1 ± 4.1 38.8 × 29.1
Range of isolate means 33.9-58.2 × 24.2-48.5 33.9-48.5 × 24.2 -33.9
L/B ratio 1.3 ± 0.1 1.4 ± 0.15 1.3
Oogonia
Mean diameter 26.5 ± 2.3 27.7 ± 1.7 21.5
Diameter range 20.9-32.1 32.6-25.5
Oospore
Mean diameter 23.6 ± 2.2 24.7 ± 1.9 18.8
Diameter range 18.8-27.9 22.1-29.8
Antheridia
L × B mean 11.2 ± 1.8 × 12.3 ± 1.4 17±1.6 × 12.8±1.3 11.5 × 10.0
L × B range 17.3-7.5 × 16.5-9.3 14.51-8.1 × 14.5-10.4
Chlamydospore
Mean diameter 31.9 ± 4.6 32.8 ± 4.2 29.1
Diameter range 24.2-43.6 29.1-38.8
Avg. growth rate on PDA (250C, mm day−1) 6.3 ± 1.5 4.9 ± 0.5 7.6
Min 3.6 3.5
Max 11.8 5.8
Avg. growth rate on V8 (250C, mm day−1) 10.4 ± 2.4 6.5 ± 0.8 12.7
Min 5.6 3.6
Max 17.1 7.7
* Spore dimension given in µm.
64 Phytophthora spp. on Citrus
Fig. 4. Restriction profiles of Phytophthora species isolates ob-
tained after digestion of ITS4/ ITS6 amplification products
(a) with MspI (b), AluI (c) and RsaI (d). Lanes 1-10, P. nicoti-
anae isolates (NRCPh 17, 18, 51, 52, 62, 63, 70, 71, 90, 98); lane
11, P. citrophthora isolate (NRCPh 97); lanes 12-18, P. palmi-
vora isolates (NRCPh 20, 21, 26, 28, 40, 92, 100). M, 1 Kb lad-
der, M’, 50 bp ladder; molecular weight in bp are indicated
adjacent to the ladder.
(nos. NRCPh- 20, 21, 26, 28, 40 and 92) and P. citroph-
thora isolate (NRCPh- 97) no products were observed. The
primer set Pal1s ⁄ Pal2a was able to amplify a unique DNA
fragment of expected length (648 bp) only in P. palmivo-
ra samples (isolate nos. NRCPh- 20, 21, 40) (Fig. 5b). No
products were observed in case of P. nicotianae isolates
(NRCPh- 18, 52, -62, -70, 90, 98) and P. citrophthora isolate
(NRCPh- 97).
Sequencing and phylogenetic analysis. The sequences
of the entire rDNA ITS (ITS1-5.8S-ITS2) regions, together
with short termini from large and small subunit genes,
were obtained from 39 Phytophthora spp. isolates and
were submitted in the GenBank database with accession
numbers as described in Table 3. The BLAST searches
confirmed identification of the sequences to P. nicotianae,
P. palmivora and P. citrophthora isolates. The PCR ampli-
cons for β-tubulin (β-tub) gene region, elongation factor-
1α (EF-1α) gene region, and cytochrome oxidase-II gene
region obtained from all the isolates for the respective
gene fragments were of expected sizes (data not shown)
and some of the representative sequences were submitted
Journal of Plant Pathology (2016), 98 (1), 55-69
Fig. 5. Agarose gels showing amplification products of Phy-
tophthora using species-specific primer pairs. PCR prod-
ucts obtained with P. nicotianae-specific primers PNIC 1/
PNIC 2 (a). P. nicotianae isolates: lane 3 (NRCPh18), lanes
8-10 (NRCPh- 52, -62,-70). P. palmivora isolates: lanes 1-2
(NRCPh-20,-21), lanes 4-7 (NRCPh-26,28,40,92); P. citrophtho-
ra isolate: lane 11 (NRCPh-97). PCR products obtained with
P. palmivora -specific primers Pal1s/Pal2a (b). P. palmivora
isolates: lanes 7-9 (NRCPh-20,-21, -40); P. nicotianae isolates:
lanes 1-6 (NRCPh- 18, 52, 62, 70, 90, 98); P. citrophthora iso-
late: lane 11 (NRCPh-97). M, 1 Kb ladder.
in the GenBank database (Table 3). BLAST searches also
indicated the relatedness of the isolates to P. nicotianae,
P. palmivora and P. citrophthora isolates with respect to
these loci.
Phylogenetic analyses were performed as described pre-
viously for the sequences of ITS, β-tub, EF-1α, and cox2.
The neighbour-joining (NJ) trees generated, revealed
diversity in sequences of all four loci (i.e. ITS, β-tub,
EF-1α and Cox2), it formed 3 major clades i.e. P. nicoti-
anae, P. citrophthora and P. palmivora, with Pythium sp.
as an outgroup (Fig. 6). Potentially all four loci clustered
the isolates in to three distinct clades together with other
GenBank accessions. Fascinatingly, intra-specific differ-
ences among the Indian P. palmivora isolates were seen in
the β-tubulin gene tree (Fig. 6).
DISCUSSION
Phytophthora spp. were isolated from all major citrus
growing regions of India surveyed in this study and the
dominant species in terms of isolate numbers was P. ni-
cotianae (80%) followed by P. palmivora (19%) and P.
citrophthora (1%). P. nicotianae, a destructive pathogen
widely isolated in tropical and warm temperate regions,
was first described in 1896 by van Breda de Haan from
an isolate collected from tobacco plants in Indonesia, but
unfortunately, the original culture was contaminated with
a Pythium species (Erwin and Ribeiro, 1996). The patho-
gen was re-isolated in India from castor bean and named
Phylophthora parasitica (Dastur, 1913), however, according
Journal of Plant Pathology (2016), 98 (1), 55-69 Das et al. 65

Fig. 6. Phylogenetic analysis of Phytophthora species on citrus using rDNA internal transcribed spacer (ITS) and partial sequences
of β-tubulin (β-tub), translation elongation factor 1 alpha (EF-1α) and region containing mitochondrial cytochrome c oxidase
subunit 2 (cox2). Neighbor-joining phylogeny based on nucleotide divergences estimated according to the Kimura 2- parameter
model implemented in MEGA 5. Numbers on nodes are the percent frequency with which a cluster appears in a bootstrap test of
1000 runs. ▲, Phytophthora species isolates from present study. Details of isolates elucidated in Table 3 and 4.

to the International Code of Botanical Nomenclature,
the original description has priority (Waterhouse, 1963).
In 1993 Hall re-described the species under the name P.
nicotianae (Hall, 1993) and this is the name used in this
paper, though P. parasitica still continues to appear in lit-
erature. Earlier several workers reported the presence of P.
nicotianae in citrus groves of India and associated it with
citrus decline (Lele and Kapoor, 1982; Sohi, 1982; Naqvi,
2006; Das et al., 2011) mainly on the basis of isolation on
selective media and their morphological characters. The
present observations also confirm previous reports of P.
nicotianae being the predominant species in citrus planta-
tions of India.
Only one isolate of P. citrophthora could be isolated and
this was from Sikkim state in North-East India, having rel-
atively wet foggy winters (average annual temperature ca.
18°C) compared to other parts of the country. It indicates
P. citrophthora is not a serious problem on citrus trunks
and roots in warm humid tropical (states of Maharashtra
and Andhra Pradesh) and subtropical areas (Punjab state)
as noted earlier in orange growing areas of Florida, USA
(Graham et al., 1998) and lower Rio Grande Valley of
Texas, USA (Kunta et al., 2007). Previously Naqvi (2006)
only on the basis of growth and morphological characters,
reported occurrence of P. citrophthora from tropical warm
humid citrus growing areas of central India, but during
our present survey not a single isolate of P. citrophthora
could be isolated from that region even from samples
collected during winter months (data not shown). It was
reported in California (which has a Mediterranean-like
climate) that P. citrophthora is active during the fall-winter
and spring but not during summer conversely P. nicotianae
is most active during summer (Dirac et al., 2003).
P. palmivora has been reported to be highly pathogenic
to fibrous and large roots of citrus as compared to P. nico-
tianae and P. citrophthora (Zitko and Timmer, 1994; Naqvi,
2004). P. palmivora was reported earlier in central India
(Lele and Kapoor, 1982; Naqvi, 2006) and in Florida, USA
66 Phytophthora spp. on Citrus
(Zitko et al., 1991) and during the present investigation its
distribution was limited to the state of Maharashtra and it
was not detected in other citrus growing states. P. palmiv-
ora may spread faster than other two species because of its
deciduous nature sporangia that are widely disseminated,
and its outcompeting parasitic ability over P. nicotianae
and P. citrophthora. (Graham et al., 2003; Naqvi, 2004). In
India, P. palmivora infects several horticultural crops such
as palms, cocoa, black pepper and cassava besides citrus
(Anandaraj, 2012).
The majority of the Phytophthora spp. isolates were iso-
lated/obtained from rhizospheric soils and fibrous roots
compared to the isolates obtained from gummosis infected
citrus bark tissues, which may be due to sample collection
after long period post infection, sampling of old inactive
lesions that were shredded, or secondary infections that
masked the primary casual organism Phytophthora. Pres-
ence of excess polyphenols and tannins (Jung, 2009) also
inhibits isolation of Phytophthora spp. from gummosis in-
fected bark tissues.
More diversity was observed in the colony morphology
of P. nicotianae isolates than in P. palmivora on V8 agar.
A total of 7 different colony patterns were observed in
80 isolates of P. nicotianae, indicating a high level of mor-
phological diversity within the species. Diverse sporangial
shapes observed in various Phytophthora spp. isolates were
ovoid, spherical/globose, limoniform, obturbinate and el-
lipsoid. Sexual reproduction in Phytophthora is regulated
by mating-type- specific hormones (Ko, 1988) and mating
types are said to be universal, in that an A1 of one species
is compatible with A2 of another species (Bonants et al.,
2000). The mating behaviour of Phytophthora species is
very important especially in relation to the survival of the
species.
Though majority of the Phytophthora isolates were
found A1 mating type, 9 isolates of P. nicotianae were
found of A2 mating type. Previously presence of both the
mating types was reported in case of P. nicotianae in the
eastern regions of India (Guha Roy et al., 2009) on crops
like brinjal, guava, betelvine, seasame, but not from citrus.
Presence of both the mating types poses a serious threat
of natural sexual recombination and development of new
strains/races having higher pathogenic ability than the
parents in the citrus orchards. The occurrence of Al and
A2 mating types in the population of P. palmivora causing
black pod disease of cocoa in India has been established
and A2 was found to be predominant mating type in P.
palmivora population in all the districts of Kerala and Kar-
nataka states in India (Chowdappa and Chndramohanan,
1997). In the present study however, there was no A2 mat-
ing type found in P. palmivora, in contrast to P. nicotianae
where both A1 and A2 mating types were observed, which
suggests the predilections of mating types in species dif-
fers even within same host (citrus). In the present study,
of 16 isolates recovered from roots, 14 were P. nicotianae
and only two P. palmivora. Although P. nicotianae has long
Journal of Plant Pathology (2016), 98 (1), 55-69
been regarded as the main cause of citrus root rot and foot
rot diseases worldwide, studies have also shown that P.
palmivora is a far more aggressive and competitive patho-
gen under Florida conditions (Graham, 1995; Bowman et
al., 2002). Moreover presence of both the species would
pose a bigger threat with regard to the formation of natu-
ral hybrids with potential infective abilities as noted pre-
viously with the discovery of natural hybrids between P.
nicotianae and P. cactorum (Man in’t Veld et al., 1998). The
only P. citrophthora isolate (NRCPh-97) was found as A1
mating type. In general, it has been assumed that the oo-
spore stage is not a part of the life history of P. citrophtho-
ra. Sex organs do not occur in nature on citrus (Erwin and
Ribeiro, 1996). In the present study, however, NRCPh-97
produced oospore when paired with A2 mating type of P.
nicotianae on carrot agar. It has been reported earlier that
P. citrophthora only acted as inducer of oospore formation
when paired with A1 or A2 mating types of other species
(Mchau and Coffey, 1994).
Growth rate of P. nicotianae was greater than that of
P. palmivora on both V8 and PDA. Most of the isolations
were done by using selective PARPH medium. The pre-
dominance of P. nicotianae in plating assays may be result
of much faster growth of this species in comparison to P.
palmivora on PARPH medium as noted earlier by Bow-
man et al. (2007). Hence, the identification of Phytophthora
using semi-selective agar-plating may sometimes lead to
inaccurate results particularly in case of mixed infection.
It is difficult and inconvenient to identify some Phy-
tophthora species based only on morphological charac-
teristics, because the available and critical morphological
characteristics categorized for identification are very rare,
the morphological features are variable and overlapping
under different environmental conditions and the iden-
tification skills are variable. Molecular data are objective
with fewer variables and, therefore, the molecular tech-
niques are frequently used in recent years as supportive
tools for identification of fungal and fungal-like organisms
including Phytophthora spp. Molecular identification of all
the Phytophthora species isolates was carried out through
internal transcribed spacer (ITS) region of the ribosomal
DNA-restriction fragment length polymorphism (ITS-
RFLP). ITS1+5.8s+ITS2 regions of the isolates were am-
plified with a set of primers ITS6 and ITS4 and amplified
products were digested with 3 restriction enzymes (RsaI,
AluI and MspI). Three different kinds of ITS-RFLP pat-
terns were obtained typical for the 3 species - P. nicotianae,
P. palmivora and P. citrophthora. The ITS region of some
of these isolates was sequenced and deposited to NCBI
GenBank. As also demonstrated by Cooke et al. (2000b),
RFLPs of the rDNA region appear to be a quite consistent
method for species identification, especially if applied to
genera such as Phytophthora, whose identification based
on morphological characters is time-consuming and re-
quires considerable experience. This study has shown that
RFLP analysis of ITS region can be used effectively and
Journal of Plant Pathology (2016), 98 (1), 55-69 Das et al.
reliably to rapidly distinguish between the three main spe-
cies of Phytophthora involved in causing diseases in citrus
as noted earlier by Bowman et al. (2007).
P. nicotianae and P. palmivora isolates were detected in
all samples with species-specific primers PNIC1/PNIC2
and Pal1s ⁄ Pal2a. Species-specific DNA primers present
an ideal alternative to PCR-RFLP technology, especially
since the time-consuming steps of digestion and electro-
phoresis to separate fragments can be eliminated. How-
ever, the simple presence of an amplification product of
specific molecular weight does not prove its identity, as
opposed to RFLP patterns based on sequence variations.
Also, design of species-specific primers derived from ri-
bosomal ITS regions can sometimes be problematic and
cross-amplification with other closely-related species is
often observed (Schubert et al., 1999; Grote et al., 2002).
During the present study, it was observed that shifting
to a more stringent annealing temperature for both the
primer pairs helped to increase the detection specificity
(data not shown). We are now working on a multiplex
assay using both pairs of primers to detect the presence
of P. nicotianae and P. palmivora simultaneously in the
infected host tissues, as occurrence of both the pathogens
are observed in the citrus orchards especially of Maha-
rashtra state.
It is noteworthy that sequence data from non-coding
ribosomal regions contain hypervariability and is more
problematic to align because it has no assigned reading
frame (Lutzoni et al., 2000). Moreover variability in ITS
sequences between closely related Phytophthora species is
low therefore using of ITS regions for phylogenetic infer-
ence is often uncertain (Alvarez and Wendel, 2003; Bai-
ley et al., 2003). Hence, besides ITS (non-coding region),
three more genetic regions i.e. β-tub, EF-1α and Cox2
(coding regions) were included for phylogenetic analysis
of Phytophthora isolates in the present study. The phylo-
genetic analysis based on four loci (i.e. ITS, β-tub, EF-1α
and Cox2) revealed that P. nicotianae, P. palmivora and P.
citrophthora isolates from India and other countries of the
world clearly fell into respective 3 different groups. The
phylogenetic grouping of isolates, however, did not corre-
late with their geographic origin, which corresponds with
previous study on unigene-derived microsatellite (UGMS)
marker pointing out that P. nicotianae isolates from India
were not geographically confined to a single phylogenetic
clade (Nerkar et al., 2012). Interestingly, in the β tubu-
lin tree, the P. palmivora clade shows more intra-specific
variation of the Indian genotypes than the P. nicotianae
clade possibly because inoculum of P. palmivora originate
from contaminated surface water sources external to citrus
plantings.
Our investigation, through morphological, molecular
and phylogenetic analyses of the collected pathogen iso-
lates, confirmed the presence of three Phytophthora species
in the citrus orchards of India, namely P. nicotianae and P.
palmivora (in tropical Indian mainland), and P. citrophthora
67
(in temperate hilly region), P. nicotianae being predomi-
nant. The wet monsoon period prevailing over various
parts of the country provides ideal conditions for them to
infect several economically important horticultural crops
including citrus. Additional sampling, however, from the
non-surveyed citrus areas with varied agro-ecological situ-
ations, might provide a clearer picture of different Phy-
tophthora species associated with various diseases causing
yield loss and citrus decline in India. Moreover the use of
a PCR-ITS-RFLP approach will help in the monitoring of
infections and the spread of different Phytophthora species
causing diseases in citrus and the species-specific DNA
primers would certainly aid in PCR-based diagnostic tests.
Furthermore, several ground bed and container nurseries,
apart from many commercial citrus groves, were found
infected with Phytophthora spp. The infested nurseries of
the respective regions were believed to be the hotspots for
dissemination of the pathogens to new areas and hence
need urgent quarantine attention. Use of Phytophthora-free
nursery planting material alongwith effective integrated
disease management (IDM) strategy is recommended to
check this menace and to enhance the productivity of cit-
rus in India.
ACKNOWLEDGEMENTS
We acknowledge the funding provided by Indian
Council of Agriculture Research (ICAR) under the Phy-
toFuRa outreach project to conduct this research.
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