American Journal of Plant Sciences, 2013, 4, 36-41
http://dx.doi.org/10.4236/ajps.2013.46A006 Published Online June 2013 (http://www.scirp.org/journal/ajps)
Identification of Alternaria spp. as Pathogenic on the
Native Species Terminalia australis and Salvia guaranitica
Mariana Kameniecki1,2, Eduardo R. Wright1*, Marta C. Rivera1
1Plant Pathology, School of Agronomy, University of Buenos Aires, Buenos Aires, Argentina; 2Asociación Ribera Norte, Buenos
Aires, Argentina.
Received April 16th, 2013; revised May 16th, 2013; accepted June 1st, 2013
Copyright © 2013 Mariana Kameniecki et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
This work is the result of activities included in the cooperative project between Asociación Ribera Norte (ARN) and the
Department of Plant Pathology, School of Agronomy, University of Buenos Aires. ARN is a non-governmental organi-
zation created in 1993 mainly for the management of the Reserva Ecológica Municipal Ribera Norte, a preservation
area located in San Isidro (North of Buenos Aires), within the Provincia Paranaense region. The aims were to identify
diseases in native plant species growing there. Light brown leaf spots with thin brown margins and narrow yellow halos
were observed on Terminalia australis (mean disease severity: 25%). The pathogen was isolated, inoculated on healthy
plants, reisolated from infected leaves and identified as Alternaria tenuissima. Similarly, another leaf-spot disease was
observed on Salvia guaranitica, characterized by reddish brown large spots developed from the apexes and margins
towards the centre of the leaves, reaching the veins in some cases (mean severity: 20%). Pathogen isolation, inoculation
and reisolation led to the identification of A. alternata as the causal agent of the disease. Unusual heavy rain is analyzed
as disease conductive.
Keywords: Alternaria tenuissima; Terminalia australis; A. alternata; Salvia guaranitica; Diagnosis
1. Introduction
Terminalia australis Cambess., belonging to Combreta-
ceae, is known as “palo amarillo” or “ta n i m b ú”. It is a
tree or shrub with grayish red cortex [1] and deciduous
elliptical leaves, native to the Northeast of Argentina [2],
and restricted in range to the more northerly provinces
[3]. It is traditionally used to obtain wood [4] that is yel-
low, compact, elastic, with remarkable durability [5] be-
cause of the astringent properties of boiled leaves [6].
This species is also used as an antiseptic agent, as it was
shown to possess antifungal properties against species of
Aspergillus and Candida [7,8].
Salvia guaranitica A. St.-Hil. ex Benth. is a rhizome-
tous species belonging to the Lamiaceae [2], usually re-
ferred to as “salvia azul” [9]. It is a semi-woody peren-
nial subshrub that exhibits a bushy, somewhat open habit
with upright, branching, square, dark green stems. The
leaves are dark green, ovate, wrinkled, pointed and ligh-
tly-toothed. This species blooms from mid-summer to
fall, producing long terminal spikes with bilabiate, tu-
bular, deep blue flowers with purple-blue calyxes. It is an
ornamental and medicinal plant originally from South
Brazil, Paraguay, Uruguay and Argentina [10], tradition-
ally used in Latin America as sedative [11]. Sedative and
hypnotic properties of the ethanolic extracts [12] have
been associated with the presence of high concentrations
of the flavonoid cirsiliol and caffeic acid ethyl ester [13].
Aerial parts also contain essential oils [9,14]. Extracts
from this species are innocuous to honey bees [15].
Salvia guaranitica has ornamental value [16] that has
led to the evaluation of procedures for its propagation
and domestication [17]. T. australis is also considered to
have ornamental characteristics [18]. T. australis has
been recorded in the provinces of Buenos Aires, Corrien-
tes, Entre Ríos y Misiones; and S. guaranitica in Buenos
Aires, Corrientes, Entre Ríos, Misiones, Salta and Tucumán
[19]. Although there is scarce information on conserva-
tion status of the flora in Argentina, both species have
been found to be included in a list of endangered trees
and shrubs in the province of Entre Ríos (Northeast of
Argentina) [20]. These species were originated in the
ecological region called Provincia Paranaense [21],
*Corresponding author.

Identification of Alternaria spp. as Pathogenic on the Native Species Terminalia australis and Salvia guaranitica 37
which is a riverside forest located at the margins of
Paraná River, later included in the Provincia Pampeana
region [22]. That environment has become threatened
because of the shift of natural areas to intensive agricul-
tural use, urbanization and dam construction. As a con-
sequence, habitat fragmentation may determine extreme
vulnerability of surviving species [23].
Asociación Ribera Norte (ARN) is a non-govern-
mental organization created in 1993 mainly for the man-
agement of the Reserva Ecológica Municipal Ribera
Norte, a preservation area located in San Isidro (North of
Buenos Aires), within the Provincia Paranaense region.
The conservation of riverside environments is in the
scope of the association. Since 1997, native plants are
grown in an orchard created there, and used to restore
natural environments and to provide specimens to be
planted in urban areas. More than 190 riverside native
species, including endemic and widely distributed ones,
are agroecologicaly cultivated [24] nowadays. Plants be-
come diseased when the ability of the cells or a plant part
to carry out one or more essential functions is interfered
by a pathogenic organism or an adverse environmental
factor [25]. Pathogens are associated with virtually all
plant species, from a diverse array of habitats. Although
we know the most about diseases of economically im-
portant plants, insufficient research has been focused on
the host-pathogen interactions in natural or unmanaged
systems outside of the crop field or forestry plantation
[26]. So as to learn more about diseases of native plants
in the area, a cooperative project was signed between
ARN and the Department of Plant Pathology, School of
Agronomy, University of Buenos Aires, in 2010. The
aims of this work were to identify diseases in plants
growing there.
2. Materials and Methods
2.1. Disease Observation, Description of
Symptoms and Isolation of Microorganisms
Monthly disease surveys began in January 2010. Samples
of diseased leaves were randomly taken from the symp-
tomatic plants, and disease severity was estimated. To
determine the causal agents of the described symptoms,
small pieces of diseased tissues were surface disinfected
by immersion in 2% (v/v) of Cl as NaOCl during 1 min-
ute, plated on PDA (pH 7), incubated at 22˚C and exam-
ined for pathogen development [27,28]. Fungal colonies
that emerged from incubated diseased leaf portions were
isolated and identified to genus.
2.2. Pathogenicity Tests and Identification of
Pathogens
Suspensions of 1 × 106 conidia ml−1 were prepared for
each isolate. Groups of 10 healthy plants of T. australis
and S. guaranitica and other 10 healthy plants with nee-
dle-punctured leaves were separately sprayed with each
inoculum suspension. Undamaged and needle-punctured
control plants were sprayed with sterilized distilled water.
All the plants were individually covered with polyethyl-
ene bags and kept in a climatic chamber under fluores-
cent light (12-hr photoperiod). The bags were removed
after 72 hr. The plants were observed regularly so as to
detect and describe the evolution of symptoms. The in-
oculated fungi were re-isolated from the leaves that de-
veloped symptoms, and the pathogens were identified to
species so as to fulfill Koch’s postulates. For fungal
identifications, the isolates were cultivated on potato
carrot agar (PCA) at 22˚C during 7 days, so as to observe
their sporulation patterns under a dissecting microscope
[29] and on malt extract agar (MA) for the observation
and measurement of conidia [30,31].
3. Results
3.1. Disease Observation, Description of
Symptoms and Isolation of Microorganisms
Leaf spots were observed on different species during
surveys carried out at the end of March 2010. Their cha-
racteristics are described below.
Terminalia australis: Pale green to chlorotic large ar-
eas appeared on the margins of the leaves, and often de-
veloped towards the main vein forming wedge shaped
lesions. The spots were finally light brown with thin
brown margins and narrow yellow halos. Leaf roll or
distortion was observed in the spotted areas (Figure 1).
The lesions covered up to 50% of the leaves. Mean se-
verity was estimated at 25%.
Salvia guaranitica: Reddish brown large spots devel-
oped from the apexes and margins towards the centre of
the leaves, reaching the veins in some cases. Smaller,
internerval, ovoid to irregular spots of the same color
Figure 1. Mature leaf lesions on T. australis.

Identification of Alternaria spp. as Pathogenic on the Native Species Terminalia australis and Salvia guaranitica 38
appeared scattered on young leaves. They measured 2.83
× 5.30 mm, mostly parallel to secondary veins (Figure 2).
Incidence was 50%, with a mean severity of 20%.
The isolates obtained from the two diseased species
produced chains of ovoid or obclavate, beaked, brown
conidia, that were multicelled, with transverse and lon-
gitudinal septa, originated from free conidiophores, and
were identified as belonging to the genus Alternaria
Nees [32,33].
3.2. Pathogenicity Tests and Identification of
Pathogens
Final symptoms were in both cases coincident with the
ones observed during the surveys. Symptoms on unda-
maged leaves appeared 14 days after those on punctured
leves. Controls remained symptomless. The inoculated
isolates were recovered from diseased leaves, thus con-
firming their pathogenicity.
Terminalia australis: Marginal lesions and also tiny
spots appeared on the leaves 20 days from inoculation
(Figure 3). Most of the foliage showed necrosis and loss
of turgency within two weeks. Severely affected leaves
dropped-off in most cases. The isolate had olive-buff
hyphae, showed a pattern of sporulation typical of Group
1 [29] and produced single conidial chains of short to
moderate length during the first week of growth on PCA
(Figure 4). Air mycelium was loose, pale gray in the
center of the plates on MA. Conidia measured 12.5 - 37.5
(24.0 µm) × 7.5 - 12.5 (10.0 µm), with 0 - 3 septa
(mainly longitudinal) and a beak of 0 - 12.5 (4 µm).
These observations are coincident with the descriptions
for Alternaria tenuissima (Kunze) Wiltshire [30]. A
specimen of the fungal culture was deposited in the
fungal collection of the Department of Plant Pathology,
School of Agronomy, University of Buenos Aires.
Salvia guaranitica : Spots of 1-mm diameter developed
on the leaves 14 days after inoculation (Figure 5). Colo-
Figure 2. Large marginal spots on leaves of S. guaranitica.
Figure 3. Initial symptoms on T. australis.
Figure 4. Sporulation pattern of Alternaria tenuissima iso-
lated from T. australis.
Figure 5. Initial symptoms on S. guaranitica.
nies were olive brown to black. The isolate had a pattern
of sporulation that belonged to Group 4 [29], with soli-
tary branching chains that turned into low bushy groups

Identification of Alternaria spp. as Pathogenic on the Native Species Terminalia australis and Salvia guaranitica 39
of branched chains of spores (Figure 6). Conidia
measured 17.0 - 40.0 (25.0 µm) × 5.0 - 17.0 (11.0 µm),
with 0 - 4 septa (mainly longitudinal) and a beak of 0 -
7.5 (2.0 µm). The morphological characteristics of the
conidia and conidiophores fit the description of A.
alternata (Fr.) Keissl. [31]. A specimen of the fungal
culture was deposited in the fungal collection of the De-
partment of Plant Pathology, School of Agronomy, Uni-
versity of Buenos Aires.
4. Discussion
The pathogenic species identified in this work show
cosmopolitan distribution. A total of 336 fungus-host
combinations, and 568 records of A tenuissima and its
synonyms can be found in the literature. Similarly, there
are 914 fungus-host combinations, and 1496 records for
A. alternata around the world [34]. No citations have
been found on T. australis and S. guaranitica; only A.
alternata on S. nemorosa in Poland [35, cited by 34]. As
a result of this work, A. tenuissima and A. alternata are
reported on T. australis and S. guaranitica, respectively,
as causal agents of leaf spots. To our knowledge, it is the
first report of the pathogens on these species, and might
be the first pathogens identified on them.
The period of incubation differed between the two
diseases; 20 and 14 days for the pathosystems T. aus-
tralis-A. tenuissima and S. guaranitica-A. alternata, re-
spectively. Wounds accelerated symptom appeareance by
two weeks for both pathologies. Local wounds are known
to increase infection by all Alternaria pathogens [36]. How-
ever, foliage injury cannot be considered a prerequisite
for fungal penetration for these diseases. In fact, both
pathogens succeeded to infect unwounded leaves, al-
though with extended incubation period when compared
with the infection on punctured foliage.
It is generally accepted that vigorously growing plants
are more resistant to infection by Alternaria pathogens
than poorly developing ones, and that soil moisture is a
major factor affecting the conditions of growth [36].
Figure 6. Sporulation pattern of Alternaria alternata iso-
lated from S. guaranitica.
Both diseases identified in this work were observed after
unusual rainy conditions that occurred in February 2010,
when accumulated rain reached 419.6 mm. Consequently,
soil moisture should be considered as conductive for
disease development in these cases. In addition, rain may
have facilitated spore germination and penetration by
supplying free moisture on leaves or by increasing rela-
tive humidity [37]. Alternaria spores are dispersed by
wind currents and are eventually deposited on the surface
of susceptible tissues [38]. The germination of spores
under high humidity conditions may take only few hours
[39].
Our results constitute the first step in the management
of plant diseases, that is the identification of their
ethiology and the estimation of damages. Infectious dis-
ease is a major causal factor in the demography of human,
plant and animal populations [40]. An intensive work has
to be done in order to identify the causal agents of new
diseases on the studied hosts or other ones, and to
understand the conducive factors, so as to evaluate tools
to preserve native species.
5. Acknowledgements
This work was supported by the University of Buenos
Aires (FAUBA).
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