Identification and characterization of

Pythium graminicola, causal agent of

kikuyu yellows in Argentina

Pablo E. Grijalba, Hemilse E. Palmucci &

Eduardo Guillin

Tropical Plant Pathology

e-ISSN 1983-2052

Trop. plant pathol.

DOI 10.1007/s40858-017-0149-1

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 Author's personal copy
Trop. plant pathol.
DOI 10.1007/s40858-017-0149-1
ORIGINAL ARTICLE
Identification and characterization of Pythium graminicola, causal
agent of kikuyu yellows in Argentina
Pablo E. Grijalba 1 & Hemilse E. Palmucci 1 & Eduardo Guillin 2
Received: 3 May 2016 / Accepted: 20 March 2017
# Sociedade Brasileira de Fitopatologia 2017
Abstract Kikuyu grass (Pennisetum clandestinum) is a peren-
nial plant that belongs to the Poaceae family. In Argentina, it is
used in warm areas as tropical pasture, forage and grass in gar-
dens and sports fields. Species of Pythium have a cosmopolitan
distribution and are important pathogens of a wide range of plant
hosts. In the spring of 2009, a new disease affected areas culti-
vated with kikuyu grass in different parks located near the city of
Buenos Aires. The symptoms were an evident chlorosis of
leaves, which got circular as the affected area expanded, followed
by severely rotten roots and blighting. The aim of this study was
to identify the causal agent of kikuyu yellows in Argentina. An
oomycete was consistently isolated from diseased tissues. The
pathogen was identified as Pythium graminicola based on cul-
tural characteristics and the morphology of vegetative and repro-
ductive structures. Phylogenetic analyses were performed based
on combined sequence datasets of the internal transcribed spacer
of the nuclear rDNA and the beta tubulin gene. Pathogenicity
tests were carried out and Koch’s postulates were fulfilled. This is
the first report of P. graminicola causing kikuyu yellows in
Argentina and in the world.
Section Editor: Meike Piepenbring
Electronic supplementary material The online version of this article
(doi:10.1007/s40858-017-0149-1) contains supplementary material,
which is available to authorized users.
* Pablo E. Grijalba
grijalba@agro.uba.ar
1
Cátedra de Fitopatología, Departamento de Producción Vegetal,
Facultad de Agronomía, Universidad de Buenos Aires, Avenida San
Martín 4453 (1417), CABA, Argentina
2
Instituto de Genética Favret, INTA Castelar, Buenos Aires, Argentina
Keywords Pythium graminicola . Kikuyu yellows .
Pennisetum clandestinum . ITS region . Beta tubulin region .
Phylogenetic analyses
Introduction
Kikuyu grass (Pennisetum clandestinum Hochst. Ex Chiov) is an
aggressive perennial plant species of the Poaceae family (Richard
et al. 2005). It spreads by underground rhizomes and long run-
ners above ground, and produces seeds through sexual reproduc-
tion. It is native from the Eastern Africa highlands, but has been
spread worldwide for forage and soil conservation purposes. In
well-managed situations, kikuyu grass does not generally spread
very far, but is highly tolerant to grazing and mowing, and can
invade poorly managed plantations. Kikuyu grass also readily
overruns natural vegetation with resultant loss of biodiversity,
as has occurred in Australia, New Zealand, South Africa,
Hawaii and the Galapagos. As a consequence, kikuyu grass is
listed as a Federal Noxious Weed in the USA (CABI 2015). In
Argentina, kikuyu grass is used in warm areas as tropical pasture,
forage and grass in gardens and sports fields.
In the spring of 2009, a new disease affected areas cultivated
with kikuyu grass in different parks located near Buenos Aires
city. Symptoms were stable across the affected area, and concur-
rent with those previously described for kikuyu yellows: an ev-
ident chlorosis of lower leaves, turning circular as the affected
area expanded, severely rotten roots, poor plant development and
blighting (Fig. 1 a, b). The disease developed at high tempera-
tures in spring and summer, which is the plant-growing period.
Kikuyu yellows is currently considered the most prevalent dis-
ease for this particular species, and was originally described in
Queensland, Australia. Verruculvus flavofaciens (P. Wong &
M.W. Dick) (Straminipila, Oomycota, Saprolegniomycetes,
Saprolegniales, Verrucalvaceae) was first identified as its causal
 Author's personal copy
a b
c d
e f
g h
Fig. 1 Morphological characterization of Pythium graminicola
associated with kikuyu yellows in Argentina. a, b Symptoms of
chlorosis in Pennisetum clandestinum. c Appressoria. Bar =30 μm. d
Toruliod sporangia. Bar =14 μm. e, f, g, h Oogonia and antheridia. Bar
=14 μm
agent. As an oomycete, this pathogen thrives in the presence of
high soil moisture (Dick et al. 1984; Thines et al. 2015).
Pythium is another oomycete, or water mold, also favored
by high soil moisture. A number of Pythium species can cause
turf diseases. Pythium is usually a seedling disease, but can
also be seen on adult turf. Affected seedlings are water soaked,
stunted, become wilted and withered and die. The disease is
promoted by warm, humid conditions in conjunction with wet
soil. Root and crown rot may occur wherever excessive mois-
ture is kept in the soil profile due to inadequate drainage.
Pythium blight is a leaf infection that creates water-soaked
looking patches; leaves in the patch may ‘stick’ together and
white mycelium may be seen in the morning or in periods of
high humidity. The infection and destruction process can be
very fast (Bransgrove 2015; Richard et al. 2005).
The genus Pythium is one of the largest oomycete genus and
consists of more than 160 recognized species, which are isolated
from different regions worldwide (Van der Plaats-Niterink 1981;
Dick 1990; Lévesque and Cock 2004; Paul et al. 2006; Bala et al.
2010; Robideau et al. 2011). Highly pathogenic species exist
Trop. plant pathol.
within this genus, such as P. aphanidermatum (Edson) Fitzp or
P. graminicola Subramaniam, both with an infecting temperature
threshold of 30 °C or higher (Van der Plaats-Niterink 1981). The
taxonomy of the genus Pythium is mainly based on morpholog-
ical descriptions and the keys provided by Middleton (1943),
Waterhouse (1968) and Van der Plaats-Niterink (1981).
However, morphological observations are now being supple-
mented with molecular information. The internal transcribed
spacer flanking the 5.8S ribosomal nuclear DNA (ITS) has be-
come a useful tool in fungal taxonomy and is currently being
utilized to identify different species (Singh et al. 2003; Paul 2001;
Lévesque and Cock 2004; Nechwatal et al. 2005). The present
study has been carried out with the main goal of identifying the
causal agent of the disease affecting kikuyu grass (Pennisetum
clandestinum) in Argentina.
Materials and methods
Samples of diseased plants were collected from parks near Buenos
Aires city during the early summer of 2010. They were stored in
plastic containers with ice at approximately 10 °C, transported to
the plant pathology laboratory (Universidad de Buenos Aires) and
processed within 48 h to obtain pure isolates. Root and rhizome
sections were washed with tap water. Small pieces of diseased
tissue were superficially disinfested with 1% NaCl for 2 min and
subsequently washed twice with sterile distilled water. The pieces
were placed on potato dextrose agar (PDA), corn meal agar
(CMA), and V8 juice agar media (V8A) with pimaricin, ampicilin,
rifampicin and pentachloronitrobenzene-hymexazol (PARPH),
and incubated at 22 ± 2 °C. Two days later, hyphal tips reaching
the surface of the medium were transferred to fresh V8A or CMA
media without inhibitors for further purification and identification,
and maintained on V8A slants. After seven days of incubation,
five colonies with cultural and morphological features of a
peronosporomycete were transferred to PDA and V8A. One col-
ony (AKIK114) was characterized using both morphological and
molecular characters, and subsequently utilized to carry out path-
ogenicity tests.
Morphological characteristics were observed on grass blade
cultures (Abad et al. 1994; Waterhouse 1968). Grass blades were
placed on CMA inoculated with the Pythium isolate. After incu-
bation for 24 h at 25 °C, the colonized blades were transferred to
10 mL of autoclaved distilled water in a 9 cm Petri dish. Thirty
oogonia were selected randomly, and the diameter of oogonia and
oospores and the number of antheridia per oogonium were mea-
sured under the microscope (Kageyama et al. 2005).
Morphological identification was based on Van Der Plaats-
Niterink (1981). Cardinal temperatures were determined on
PDA. Petri dishes were inoculated in the center with a 7 mm
diameter agar plug. After 24 h two lines were drawn perpendicu-
larly to each other at the bottom of the Petri dish, which intersected
at the place of the inoculum, and colony margins were marked on
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Trop. plant pathol.
these lines. Petri dishes were subsequently incubated in the dark at
0, 5, 10, 15, 20, 25, 30 and 35 °C. After 24 and 48 h the radial
growth was determined in all four directions. If growth stopped
before the edge of the Petri dish was reached, the culture was
returned to 20 °C to check if growth could resume and if the
culture was still viable.
Pathogenicity tests
Pathogenicity tests were carried out on commercial sods or
pieces of kikuyu grass of 0.60 × 0.40 m, using the inoculum
layer technique (Schmitthenner et al. 1994). A 9 cm disc of
PDA with 10-day-old mycelium was cut and mixed with the soil
around the roots and rhizomes of four sods (one colony per sod).
The mixture was placed in trays, covered with plastic bags and
incubated at 25 ± 2 °C and 100% relative humidity for 72 h, and
then transferred to a greenhouse with natural light and soil mois-
ture between field capacity and saturated water content for ap-
proximately 30 days, when disease symptoms were recorded.
Control plants were inoculated with agar discs that did not con-
tain the oomycete.
Molecular analysis
DNA extraction was made from a pellet containing mycelium
and reproductive structures of the oomycete grown 7 to 10 days
in PDA, using the Nucleon PhytoPure Genomic DNA Extraction
kit (GE Healthcare) according to the manufacturer’s instructions.
Two nuclear loci were selected for sequence characterization, the
ITS rDNA region (primers ITS4 and ITS5; White et al. 1990)
and a portion of the beta tubulin (β-tub) gene (Villa et al. 2006).
Amplification was carried out in 50 μL of PCR Buffer
(Boehringer Mannheim) in the presence of 150 ng of each prim-
er, 200 mM of each dNTP (Boehringer Mannheim), 10 ng of
DNA and 2 U of Taq polymerase (Boehringe Mannheim).
Amplification of the sequencing template was carried out in a
DNA thermal cycler (Perkin-Elmer Cetus) with a cycling profile
of pre-PCR at 94 °C for 5 min, followed by 40 cycles of dena-
turation at 94 °C for 1 min, 1 min primer annealing at 55 °C for
ITS and 63 °C for β-tub and elongation at 72 °C for 2 min, with a
7 min extension at 72 °C after the final cycle. To check the
presence of PCR products, 5 mL of the PCR reaction mixture
was electrophoresed on 1% (w/v) agarose gels run at 100 V for
1 h in tris-acetate buffer, stained with ethidium bromide, and
visualized under UV light. PCR products were desalted with
the ExoSAP-IT for PCR Product Cleanup kit (Affymetrix) ac-
cording to the manufacturer’s instructions. Desalted amplicons
were sequenced at the Servicio de Genotipificación y
Secuenciación, Instituto de Biotecnología del Centro Nacional
de Investigaciones Agropecuarias (INTA, Buenos Aires). In or-
der to minimize sequencing errors, both strands were sequenced.
Dataset assembly and sequence analysis
A consensus sequence for both the ITS and β-tub regions was
inferred from forward and reverse sequences using GeneTool Life
1.0 (Layon 2000). Such sequences were first compared against the
non-redundant database at the NCBI using the Basic Local
Alignment Search Tool (BLAST) program (Altschul et al.
1997), with the Megablast algorithm (highly stringent conditions).
Sequences from those searches were retrieved and stored to devel-
op the working datasets. Alignements for each locus were carried
out using Clustal W as implemented in MEGA 6.0 (Tamura et al.
2013). Between groups and within group uncorrected genetic dis-
tances were also obtained using MEGA 6.0.
Bayesian phylogenetic analysis
The genealogical relationships between the local kikuyu yellows
strain and a number of Pythium species retrieved from the
BLAST search were inferred by a Bayesian phylogenetic analy-
sis. For each gene region, the Akaike Information Criterion
(Akaike 1974) as implemented in MrModeltest v.2.3 (Nylander
et al. 2004) indicated the best-fit model of molecular evolution
[HKY + G for the ITS locus (dataset 1) and HKY + G + I for the
β-tub locus (dataset 2)]. Datasets 1 and 2 included 48 sequences,
with nine represented species in each one (Suppl. files 1–2).
Dataset 3 is a concatenated supermatrix including 28 isolates
from datasets 1 and 2 with both ITS and β-tub sequences avail-
able. All nine species are therein represented (Suppl. file 3). This
concatenated supermatrix was also best accounted for using the
HKY + G substitution model (Hasegawa et al. 1985). Bayesian
topologies for each individual locus and the concatenated
supermatrix were obtained using MrBayes v3.1.2 (Ronquist
and Huelsenbeck 2003). Analyses were run under the following
conditions: two simultaneous, independent runs of five million
generations each, one cold and four heated chains in each run; the
temperature parameter was set to 0.25 and the branch length prior
mean was set to 0.01. Trees were sampled once every 5000
generations. The first 25% trees were discarded as burn in; aver-
age standard deviation of split frequencies at the end was below
0.01. The selected settings ensured sufficient sampling of poste-
rior probabilities (effective sample size over 400 for all statistics)
as assessed using Tracer 1.5 (Rambaut and Drummond 2009). A
50% majority rule consensus tree was obtained to present results.
Trees were visualized using FigTree v.1.4.2 (Rambaut 2009).
Results and discussion
Morphological studies
Five isolates of the pathogen associated with the disease had the
same growth pattern and morphological features: white colonies
on CMA forming poorly developed aerial mycelium, on PDA
 Author's personal copy
submerged with fine radiate pattern colonies. The isolate
AKIK114 was used for the morphological characterization and
is kept in the World Oomycete Genetic Resource Collection
(WOC) with the Number P19707 and in the culture collection
of the Phytopathology Chair of the Facultad de Agronomía de
Buenos Aires (FAUBA). Coenocytic hyphae and hyphal swell-
ings were observed in microscopic slides. Appressoria are
subspherical or irregular (Fig. 1c) and sporangia were not ob-
served on solid agar, but readily produced in water, showing
toruliod sporangia (Fig. 1d). Oogonia were produced abundantly
in single culture, strictly globose, smooth-walled, terminally and
intercalary borne. Antheridia were usually monoclinous, often
also diclinous, 1–6 per oogonia. Oospores were single, plerotic,
completely filling the oogonia (Fig. 1 e, f, g, h). Cardinal tem-
peratures were 15 °C and 35 °C with the maximum growth rate
at 30 °C on PDA. Measurements of oogonia were 20–25 μm
(avg. 23.1 μm) diam. All these characteristics are consistent with
those described for P. graminicola (Van der Plaats-Niterink
1981).
Molecular characterization
BLAST comparisons revealed high similarity between the ITS
and β-tub sequences of the Argentinean kikuyu yellows agent
and those from three different species of Pythium, namely
Pythium graminicola, P. periilum and P. tardicrescens (inter-
group genetic distances of p = 0.00). Overall genetic distance
for the ITS dataset was 0.03; between-group (species) distances
for the ITS locus ranged from 0.00 to 0.07, which contrasted to
within-group distances of 0.00–0.01. Additionally, and in order
to further tell P. graminicola apart from P. perilium and
P. tardicrescens, we attempted the utilization of the tub locus.
Analysis of the tub locus yielded an overall uncorrected genetic
distance of 0.05 for the entire dataset; between-group distances
ranged from 0.00 to 0.10 and again, within-group distances
ranged between 0.00 and 0.01.
Phylogenetic analysis
Bayesian phylogenetic trees showed that sequences from
P. graminicola, P. periilum and P. tardicrescens clustered to-
gether into a single clade (posterior probability, pp = 1.0) in all
three datasets (Fig. 2a–c). The Argentinean kikuyu yellows
isolate is located within that cluster. Other related species
(P. torulosum, P. sulcatum, P. volutum and P. vanterpolii) clus-
tered apart, each forming independent clusters, with pp = 1.0.
Isolates identified as belonging to P. inflatum are not clearly
defined with these datasets.
Lévesque and Cock (2004) conducted a broad molecular
phylogenetic analysis in which they compared sequences
from 116 species of Pythium retrieved from GenBank, and
Trop. plant pathol.
c o n c l u d e d t h a t P. p e r i i l u m i s s y n on y m o u s w i t h
P. graminicola, which presents identical ITS sequences and
morphological similarities. The main differences are the larger
oogonia, (22.3 μm on average in P. graminicola and 20.0 μm
on average in P. periilum) and the presence of strictly filamen-
tous elements in the sporangia of P. periilum. Identical results
were reported by Lodhi (2007), considering ITS and LSU
analyses. Results herein reported, based on the ITS and tub
loci, agree with those from Lodhi (2007) and Lévesque and
Cock (2004). Pythium periilum was described by Drechsler
(1930) and was isolated in Algeria, Australia, Florida, India
and Togo (CBS 2016; Van der Plaats-Niterink 1981). P.
tardiscrescens was originally isolated from diseased wheat
roots in Canada. It was later recorded on grass in England
and the USA (Van der Plaats-Niterink 1981), and in Brazil
in 2004 (Baptista et al. 2004). P. tradiscrescens presents
aplerotic oospores while in P. graminicola the oospores are
plerotic; also, the daily growth rate is slow in P.
tradiscrescens. In the present report, no sequence-based dif-
ference was detected amongst P. tardicrescens, P. graminicola
and P. periilum.
Pathogenicity tests
Leaf chlorosis symptoms were observed 30 days after inocu-
lation. The disease progressed and caused yellowish brown
roots with incipient rot. Control plants remained symptomless.
Koch’s postulates were confirmed by re-isolating the same
microorganism from diseased plants.
Verrucalvus flavofaciens causes a disease in kikuyu grass
with the same symptoms described above and shares many
characteristics with the isolates obtained in this research,
which probably caused its previous misidentification based
only on disease symptoms and morphological characteristics
(Grijalba and Palmucci 2011). In this paper, the results of two
molecular markers and Bayesian phylogenetic analysis, along
with corresponding pathogeniticty tests, allowed the correct
identification of Pythium graminicola as the causal agent of
Argentinean kikuyu yellows.
Dick et al. (1984) indicated that the oospore of Verrucalvus
flavofaciens has noteworthy features: it is plerotic even into
the oogonial neck, the ooplast appears to develop a condensed
core, and the relative thicknesses of the verrucated oospores
appear to differ from those of other oomycetes. It would have
beeen informative to include sequences of Verrucalvus
flavofaciens in our phylogenetic analyses. However, such in-
formation is not available in public databases. Therefore, no
molecular differentiations could be made between
V. flavofaciens and P. graminicola.
P. graminicola was originally described from wheat
roots in India. It has been recorded several times in
 Author's personal copy
Trop. plant pathol.
Fig. 2 Bayesian phylogenetic trees reflecting the relationship between
the Argentinean kikuyo yellows agent and Pythium species, as retrieved
from GenBank. a Phylogenetic tree based on the ITS locus; b
grasses, especially those belonging to the Poaceae fam-
ily (http://nt.ars-grin.gov/fungaldatabases/), on which it
can be an important pathogen, and has a worldwide
occurrence (Van der Plaats-Niterink 1981; Waterhouse
and Waterston 1964). In Argentina, P. graminicola was
isolated by Frezzi (1956) from Maranta arundinacea
with stunting and chlorosis due to root rot. Years later,
Phylogenetic tree based on the β-tub locus. c Phylogenetic tree based
on the ITS-β-tub concatenated supermatrix. Numbers above the
branches indicate posterior probabilities
it was reported as a pathogen of Saccharum officinarum
(sugarcane) (Nome et al. 2012).
The combination of morphological and cultural character-
istics and phylogenetic analysis showed that isolate
AKIK114, obtained from Pennisetum clandestinum samples
symptomatic for kikuyu yellows, is a member of the species
Pythium graminicola. To our knowledge, this is the first report
Author's personal copy
Trop. plant pathol.
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Trop. plant pathol.
of P. graminicola causing kikuyu yellows in Argentina and in
the world.
Acknowledgements The authors would like to thank Dr. Gloria Abad
(USDA-APHIS-PPQ Center for Plant Health Science & Technology).
This research would have been impossible without her great knowledge.
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