Diversity of Ascomycetes at the Potato Interface: New Devastating Fungal Pathogens Posing Threat to Potato Farming
Sayanika D. Waikhom,
Wakambam M. Singh,
Narayan C. Talukdar
Potato cultivation is hampered by a host of factors among
which fungal diseases play a leading role. Nevertheless, our knowledge on phytofungal
diversity inciting diseases at the early developmental stages of potato is limited
at present to some few traditionally recognized pathotypes. In this present
study, potato farms were surveyed for two and a half years and predominant fungi
causing disease at the early stages of potato development were identified using
standard taxonomic monographs and ribosomal DNA locus. It was found that Ascomycetes
viz. Colletotrichum gloeosporioides, Cochliobolus lunatus, Aspergillus
terreus, Aspergillus fumigatus, Nigrospora oryzae, Fusarium culmorum,
Pestalotiopsis vismiae, Phomopsis asparagi, Dothiorella gregaria, Didymella
cucurbitacearum, Stagonosporopsis cucurbitacearum and Penicillium citrinum
incite diseases of potato at the early developmental stages. This study
provide a comprehensive overview of the genetic diversity of Ascomycetes thriving
on potato plants and evidence that Aspergillus species are harmful pathogens
causing loses to potato cultivation.
to cite this article:
Bengyella Louis, Sayanika D. Waikhom, Wakambam M. Singh, Narayan C. Talukdar and Pranab Roy, 2014. Diversity of Ascomycetes at the Potato Interface: New Devastating Fungal Pathogens Posing Threat to Potato Farming. Plant Pathology Journal, 13: 18-27.
Received: September 02, 2013;
Accepted: November 04, 2013;
Published: March 18, 2014
The continuing decrease in field surveillance program and mining of emergent
pathogens has led to our poor understanding of Ascomycetes diversity and the
threat they pose in potato (Solanum tuberosum L.) fields. Usually, the
use of pathogen contaminated tubers for seed favour diseases at the early developmental
stages of potato. Congruently, most of the organisms causing disease at the
early developmental stages go unidentified because of incorrect diagnosis since
all parts of the plant are usually affected with rare characteristic symptoms.
Common blight pathogens of potato are Phytophthora infestans (causing
late blight diseases), Alternaria solani (causing early blight disease),
Verticillium spp. (causing early dying disease), Helminthosporium
solani (causing silver scab disease), Colletotrichum coccodes (causing
black dot disease), Synchytrium endobioticum (causing wart disease),
Fusarium spp. (causing Fusarium wilt disease) and Alternaria
alternata (causing brown spot and black pit disease). Although, these pathogens
causes early blight diseases, the term early-blight is misleading
because these diseases are often observed on older leaves at the late stages
of development not at the early developmental stages of the plant. Based on
this pre-existing data for early blight diseases, farmers, especially those
of Burdwan, West Bengal, India, subjectively apply fungicides such as mancozeb,
copper oxychlorides and mancozebmetalaxyl to control potato diseases (Bengyella
et al., 2012). This subjective approach is usually based on the assumption
that common phytofungi of potato prevails. In most cases, this control measure
fails leading to substantial loses and thus; indebting farmers. Addressing this
problem requires a strategic surveillance of potato field and identifying recurring
phytofungi so that appropriate control measure can be established. In this study,
potato fields were surveyed for phytofungi causing diseases at the early developmental
stages of potato. Here, it is observed that Ascomycetes poses severe threat
to potato farming.
MATERIALS AND METHODS
Study area, sampling and identification: The study was performed in
potato farms of Burdwan District (23°14N, 87°51E, altitude 150 m,
102.1 km from Kolkata), West Bengal, India, during winter 2010 to 2012. The
area receives mean annual rainfall of about 1173-1442 mm and temperature of
10-20°C during potato farming season. Only four to six weeks old potato
plants were sampled following development of true leaves. Diseased plants that
amounted to more than 5% of the sampled plot with uniform pattern of symptoms
were considered for further studies. Diseased potato leaves and stems were excised
and treated with 2% NaClO solution (2 min) and rinsed in sterile water with
three changes. The tissue pieces were aseptically plated on PDA (HiMedia®)
amended with 250 mg L-1 chloramphenicol and incubated at 25°C
in dark. Developed colonies were further purified on V8 agar medium and subsequently
identified morphologically based on standard monographs taxonomic keys. However,
non sporulating isolates that could not be mapped into any taxonomic position
but produced significant crop damage were only characterized molecularly. Microscopy
observation was performed under a phase contrast Olympus BX61 microscope couple
with DP7M184.108.40.206 software and an Olympus DP70 camera.
Pathogenicity test: Pathogenicity test were performed on three weeks
old disease free potato plants (cv. Kufri Jyoti) grown in a 7 L capacity pots
under greenhouse conditions. Autoclaved soil derived from a blend of rice stalk
vermicompost and sand (1:2% w/w) was used. The soil was amended with 1 g NPK
(1:1:1% w/w) fertilizer after one week of sprouting. A fungal suspension of
106 conidia mL-1 was sprayed on the leaves using a hand
compression sprayer. Control plants were sprayed with sterile water only. Plants
were covered with polyethylene bags (at near 100% humidity) and incubated for
four days at 20±2°C. Fungi isolates that produced disease symptoms
similar to the field symptoms were re-isolated and characterized further using
DNA phylogenetic analysis: Total genomic DNA was extracted from mycelium
mat using UltraCleanTM Microbial DNA isolation kits (Mo Bio Laboratories,
Inc., Carlsbad, CA, USA) essentially following the manufacturer protocol. The
purity of DNA was checked by the absorbance measurements at 260/280 nm using
a Shimadzu® BioSpec-nanodrop spectrophotometer. The integrity of the DNA
was confirmed by agarose gel electrophoresis. The ribosomal DNA internal spacer
region 2 was amplified using ITS4 (5-tcctccgctt attgatatgc-3) and
ITS3 (5-gcatcgatgaag aacgcagc-3) primers. The PCR mix contained
10 ng DNA, 5 μL Green GoTaq® reaction buffer (Promega®), 0.2 mM
of each deoxyribonucleoside triphosphate (dNTP), 0.2 μM of each primer
and 1.1 U of GoTaq® DNA polymerase (Promega®) in a total reaction volume
of 25 μL in triplicates (conditions: 5 min at 95°C; 38 cycles of 1
min at 94°C, 1 min annealing at 56°C, 2 min for extension and a final
5 min at 72°C). The quality of the amplification products was checked on
2% agarose gel electrophoresis. The amplicons were purified and sequenced. The
sequences have been submitted to the GenBank® nucleotide database based
on 99.5 to 100% sequence similarity and have been awarded accession numbers
(Table 1). To establish the diversity among these pathogens,
sequences were aligned in Muscle software (Edgar, 2004).
Substitution model parameters were determined based on Akaike Information Criterion,
corrected (AICc) and Bayesian Information Criterion (BIC). The phylogenetic
tree was inferred using the maximum likelihood method in MEGA5.2 software (Tamura
et al., 2011).
RESULTS AND DISCUSSION
Among the diseases recorded, only three cases of early blight caused by Alternaria
species were observed in the month of November to December (Table
1) and the conidial morphological variations are represented (Fig.
1). Alternaria blight caused by A. alternata has been reported
in India on Rumex vesicarius (Sankar et al.,
2012) and in neighbouring Pakistan (Siddiqui et
al., 2009). However, based on the literature Alternaria solani
(Fig. 1a) is herein reported for the first time to cause early
blight disease of potato in India. Symptoms under greenhouse conditions were
scattered brown spots on leaflet which often coalesced. Because the occurrence
of Alternaria blight was low within the two and a half years surveillance,
this indicated other unidentified pathogens are actively involved accounting
for substantial loses of potato plants at the early developmental stages.
Following inoculation of Colletotrichum gloeosporioides under greenhouse
condition, atypical symptoms similar to those of the field developed. No symptoms
were observed on control plants. Often, plant progressively shriveled followed
by discoloration (Fig. 2). Here, damping off and curling of
apical leaves associated with occasional black spot on decolorized leaves were
observed. This disease was encountered once in the field only in the month of
October (Table 1). Colletotrichum gloeosporioides has
been reported on Jasmimum grandiflorum in India (Sharma
et al., 2012). To our knowledge, this is the first report of
Colletotrichum gloeosporioides causing disease of S. tuberosum L.
||Diversity of Alternaria, the main causal of early blight
diseases identified in Burdwan potato farms, (a) Alternaria solani,
(b) Alternaria sp. and (c) Alternaria sp. cultured on V8 agar
||Ascomycetes causing diseases of potato at the early development
stages reveals high occurrence level in the month of November and December
|*Isolates that were characterized morphologically solely
Among all the pathotypes identified, the occurrence of Cochliobolus lunatus
(Fig. 3) was high. C. lunatus has been reported to
cause severe foliar necrosis at all stages of potato development (Bengyella
et al., 2013a).
Under field conditions Cochliobolus lunatus, Aspergillus spp.,
Colletotrichum gloeosporioides and Alternaria solani caused significant
losses of about 46-60%.
||Potato disease characterized by damping-off and occasional
black spots caused by Colletotrichum gloeosporioides
||A typical conidia morphological variations of Cochliobolus
lunatus (GenBank accession JX477595) cultured on V8 agar medium
Disease caused by Aspergillus spp. and Penicillium citrinum
were all similar in nature to those of Aspergillus terreus under greenhouse
conditions as described in Bengyella et al. (2013b).
Nonetheless, A. terreus was more virulent and symptoms developed within
three days of inoculation (Fig. 4).
||Leaf blight of potato caused by Aspergillus terreus characterized
by brown apex, spots and chlorosis
Although Aspergillus spp. and Penicillium spp. are often considered
as weak pathogen of plants (Gugnani, 2003; Pitt
and Hocking, 1997), it was found that Penicillium citrinum (Fig.
5a), Aspergillus aculeatus (Fig. 5b), Aspergillus
terreus (Fig. 6a), Aspergillus sydowi (Fig.
6b), Aspergillus fumigatus (Fig. 7a) and Aspergillus
niger (Fig. 7b) were successfull in inciting diseases
Importantly, it was observed that Didymella cucurbitacearum, Stagonosporopsis
cucurbitacearum, Diaporthe phaseolorum, Nigrospora oryzae,
Phomopsis asparagi and Gibberella zeae failed to incite diseases
under greenhouse conditions. This result suggested that these pathogens required
some abiotic or biotic factors to thrive on the plant which was excluded under
greenhouse conditions or were simply opportunistic pathogens. Furthermore, these
pathogens were associated with decomposition of plant tissues. Diseases caused
by the Fusarium sp. under greenhouse conditions were severed. For instance,
Fusarium culmorum caused stem wilting, stunting, chlorosis and leaf necrosis
(Fig. 8). The pathogenicity of the pathogen developed slowly
and disease persisted to the advanced stages of development, colonizing the
xylem vascular tissues and producing browning of stem tissues as indicated by
arrows (Fig. 8).
||(a) A head of typical Penicillium citrinum and (b)
A typical conidiophore head of Aspergillus aculeatus consisting of
a central vesicle (a) which produces a dense layer of cells called metulae
(b) which develps into phialides (c) from which conidia develops (d) and
the whole structure is supported by stipe (e) Isolates were cultured on
V8 agar medium and imaged at 1000X
||(a) A typical conidiophore head of Aspergillus terreus
characterized by a smooth columnar broom-like conidiophore and (b) Aspergillus
sydowii characterized by slightly brownish conidiophore and spherical
conidia (2.84 μm), Isolates were cultured on V8 agar medium and capture
||(a) Conidiophore head of Aspergillus fumigatus and
(b) Conidiophore head Aspergillus niger, Isolates were cultured on
V8 agar medium and capture at 1000X
||Blight disease characterized by wilting under greenhouse conditions
caused by Fusarium culmorum strain btl18IBSD GenBank accession KC937044,
Image acquired with Nikon CoolpixS6200 under greenhouse condition
According to the literature F. oxysporum variants has been reported
in India on potato (Mandhare et al., 2011),
but not F. culmorum. This is a first report demonstrating that F.
culmurum causes disease of potato in India.
To date, Dothiorella gregaria (Fig. 9) is essentially
reported to cause Dothiorella canker disease of avocado (Hartill
and Everett, 2002). In potato the pathogen caused leaf discoloration associated
with apex distortion. Often, chlorosis originated on or near the leaf apex (Fig.
||A typical conidia morphological variations of Dothiorella
gregaria (GenBank accession KC937051) cultured on V8 agar medium imaged
It was observed that the disease does not reduce the plant vigour; however,
surface discoloration could amount to 30-50% of the total leaf surface. This
observation indicated that photosynthesis is severely hampered. To our knowledge,
this is the first report of foliar disease of potato caused by Dothiorella
gregaria in India.
It is worth noting that all the pathotypes belonged to the phylum Ascomycota.
In essence Penicillium, Aspergillus and Alternaria, Cochliobolus
produced huge numbers conidia which on a putative host lead to rapid colonization;
however, does not explain the gain of new host. Primitive genes involved in
pathogenicity are enzymatic such as cellulose encoders for plant cell wall decomposition.
Meanwhile, advance pathogenicity and virulence factors are toxins. Ascomycota
such as Penicillium, Aspergillus, Alternaria, Cochliobolus,
Fusarium has been shown to produce host specific toxins (Yoder
et al., 1997; Salas et al., 1999)
and concurrently exhibit high level of virulence. Meanwhile, certain factors
such as temperature fluctuation stress associated with insects damages and drought
can readily weaken plant resistance to these emerging pathogens; thereby facilitating
fungal invasion and subsequent toxin production (McMillian
et al., 1998; Salas et al., 1999).
||Leaf discoloration of potato caused by Dothiorella gregaria
under greenhouse condition and image was acquired with Nikon CoolpixS6200
Molecular Phylogenetic analysis by Maximum
Likelihood method. The ribotype tree is drawn to scale and was inferred
by using the Maximum Likelihood method based on the General Time Reversible
(GTR+G+I) model (Nei and Kumar, 2000
are supported by 1000 bootstrap test of replicates and Aspergillus
strain btl81BSD KC937034 forms the out-group
Some of the above mention factors were absence under the greenhouse condition
and could explain why some pathotypes failed to incite disease. Importantly,
Thompson (1994) argued that Ascomycetes are excellent
ecological opportunist, suggesting in a given biota, they could
coevolved and thrived on any putative host.
Final sequence alignment matrix for 5.8S-ITS2-28S rDNA locus provided 355 information
patterns out of a total of 393 sites and 84 sites without polymorphism (21.37%).
The GTR+G+I nucleotide substitution model was used base on AICc (2801.958) and
BIC (3160.941). The rate of nucleotide transitional substitution were (A-G)
= 10.28, (C-T/U) = 10.28, (T/U-C) = 10.28 and (A-G) = 10.28. Initial tree(s)
for the heuristic search were obtained by applying the Neighbor-Joining method
to a matrix of pairwise distances estimated using the Maximum Composite Likelihood
(MCL) approach. A discrete Gamma distribution was used to model the evolutionary
rate differences among sites (5 categories (+G, parameter = 8.5974)). The rate
variation model allowed for some sites to be evolutionarily invariable ((+I),
0.0000% sites). The tree is drawn to scale, with branch lengths measured in
the number of substitutions per site (Fig. 11). The analysis
involved 27 fungal isolates identified and sequenced. All positions containing
gaps and missing data were eliminated. The percentage of trees in which the
associated isolates clustered together is shown next to the branches following
1000 bootstrap test of replicates.
More than 90% of pathogens that thrives on S. tuberosum L. under field
conditions goes identified. This is probably because fungicides are applied
sequentially in most cases at the early stages of development till harvest in
order to optimize yield. As a consequence, necrotrophs and heminecrotrophs which
are chiefly Ascomycetes are severely suppressed, favouring the detection of
biotrophs such as the Oomycetes which are specific to potato. Unequivocally,
fungicide application eliminates the episode of Ascomycetes host gains as well
as their pathogenicity evolutionary history. Base on this argument, pathogens
that have coevolved with host or switch host to adapt in their biota in a given
farming season eludes detection and characterization. Based on the ribotype
tree (Fig. 11) clade 1 was made of Pezizomycetes, Sordariomycetes
and Dothideomycetes, clade 2 was composed of Pezizomycetes and Sordariomycetes,
clade 3 was composed of Sordariomycetes and Eurotiomycetes and clade 4 was made
of Sordariomycetes, Dothideomycetes and Eurotiomycetes (Table
1). Previously, robust phylogenetic analysis revealed plant pathogens were
concentrated at the level of Sordariomycetes while animal pathogens were focused
in the Eurotiomycetes (Berbee, 2001). Our result show
changes in the pathogenicity host-gains of Ascomycota; where Pezizomycetes,
Sordariomycetes, Eurotiomycetes and Dothideomycetes are involve in inciting
diseases of potato.
It was observed that all Cochliobolus lunatus strains failed to cluster
indicating divergent evolutionary pattern. Akin to this pattern, Aspergillus
terreus and Aspergillus fumigatus failed to cluster with other Aspergilli.
Among all the identified fungi, Cochliobolus lunatus, Aspergillus
sp. and Fusarium sp. has been reported to cause disease in both plants
and animals. And as such, they represent potent dangers to many organisms and
are generally called cross-kingdom pathogens (Berbee, 2001).
Agricultural loses in potato caused by Ascomycota can sum up to be significant
to those of Oomycetes such as Phytophthora spp. if the latter is controlled
and the former is neglected. A significant damage caused by an Ascomycota is
the Great Bengal rice famine caused by Cochliobolus spp. that lead to
the loss of over 2 million lives (Scheffer, 1997). This
case demonstrates the destructive potential of Ascomycetes when uncontrolled.
An efficient integrated disease management strategy to protect crops can only
be implemented when the prevailing pathogens are identified. In this study,
newly identified pathogens of potato are Cochliobolus lunatus, Aspergillus
terreus, Colletotrichum gloeosporioides, Aspergillus fumigatus, Nigrospora
oryzae, Fusarium culmorum, Pestalotiopsis vismiae, Phomopsis asparagi, Dothiorella
gregaria, Didymella cucurbitacearum, Stagonosporopsis cucurbitacearum and
Penicillium citrinum. Future surveillance of potato farms untreated with
fungicides would supplement the current knowledge of diversity of Ascomycota
and the magnitude of damages they caused in potato farms.
This study was funded by The Academy of Sciences for Developing World (TWAS)
and Department of Biotechnology (DBT), Government of India (Program No. 3240223450).
The authors extend their gratitude to D.K. Hore, R.C. Rashmi and D.G. Momin
for proofreading the text.
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