Identification of Alternaria spp. as Pathogenic on the Native Species Terminalia australis and Salvia guaranitica

doi:10.4236/ajps.2013.46A006

Paper Menu >>

Journal Menu >>

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.

Email: *[email protected]

Received April 16th, 2013; revised May 16th, 2013; accepted June 1st, 2013

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

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).

REFERENCES

[1] A. L. Cabrera and E. H. Zardini, “Manual de la Flora de

los Alrededores de Buenos Aires,” ACME, Buenos Aires,

1978.

[2] M. J. Dimitri, “Enciclopedia Argentina de Agricultura y

Jardinería. Vol. I,” ACME, Buenos Aires, 1987.

[3] A. W. Exell, “The Combretaceae of Argentina,” Lilloa,

Vol. 5, 1940, pp. 123-130.

[4] J. N. Cardoso Marchiori, “Descriptive Wood Anatomy of

Terminalia australis Camb. (Combretaceae),” Revista do

Centro de Ciências Rurais, Vol. 16, No. 4, 1886, pp. 329-

340.

[5] M. P. Corréa, “Diccionário das Plantas Úteis do Brasil—

E das Exóticas Cultivadas. Vol. 1,” Imprensa Nacional,

Rio de Janeiro, 1926.

[6] A. Bertucci, F. Haretche, C. Olivaro and A. Vázquez,

“Phytochemical Screening of the Native Forest of Uru-

guay River,” Revista Brasileira de Farmacognosia. Bra-

zilian Journal of Pharmacognosy, Vol. 18, No. 1, 2008,

pp. 21-25.

[7] S. M. Carpano, E. D. Spegazzini, J. S. Rossi, M. T. Castro

and S. L. Debenedetti, “Antifungal Activity of Terminalia

australis,” Fitoterapia, Vol. 74, No. 3, 2003, pp. 294-297.

doi:10.1016/S0367-326X(03)00026-1

[8] L. Muschietti, M. Derita, V. Sülsen, J. D. Muñoz, G. Ferraro,

S. Zacchino and V. Martino, “In Vitro Antifungal Assay

of Traditional Argentine Medicinal Plants,” Journal of

Ethnopharmacology, Vol. 102, No. 2, 2005, pp. 233-238.

Identification of Alternaria spp. as Pathogenic on the Native Species Terminalia australis and Salvia guaranitica

doi:10.1016/j.jep.2005.06.017

[9] A. Lombardo, “Flora Montevidensis. Vol II. Gamopéta-

las,” Intendencia Municipal de Montevideo, Montevideo,

1983.

[10] C. Pereira, “Flora do Estado do Paraná,” Família Labiatae,

Leandra, Vol. 1, 1971, pp. 13-33.

[11] H. Viola, C. Wasowski, M. Marder, C. Wolfman, A. C.

Paladini and J. H. Medina, “Sedative and Hypnotic

Properties of Salvia guaranitica St. Hil. and of Its Active

Principle, Cirsiliol,” Phytomedicine, Vol. 4, No. 1, 1997,

pp. 47-62. doi:10.1016/S0944-7113(97)80027-X

[12] J. H. Medina, C. Peña, M. Levi de Stein, C. Wolfman and

A. C. Paladini, “Benzodiazepine-Like Molecules as Well

as other Ligands for the Brain Benzodiazepine Receptor

are Relative Common Constituents of Plants,” Biochemi-

cal and Biophysical Research Communications, Vol. 165,

No. 2, 1989, pp. 547-553.

doi:10.1016/S0006-291X(89)80001-4

[13] M. Mader, H. Viola, C. Wasowski, C. Wolfman, P. G.

Waterman, J. H. Medina and A. C. Paladini, “Cirsiliol

and Caffeic Acid Ethyl Ester, Isolated from Salvia guara-

nitica, are Competitive Ligands for the Central Benzodi-

azepine Receptors,” Phytomedicine, Vol. 3, No. 1, 1996,

pp. 29-31. doi:10.1016/S0944-7113(96)80006-7

[14] C. Vallverdú, R. Vila, D. Lorenzo, D. Paz, E. Dellacassa,

P. Davies, J. Villamil, F. Tomi, J. Casanova and S. Ca-

ñigueral, “Composition of the Essential Oil of Cultivated

Salvia guaranitica from Uruguay,” Flavour and Fragance

Journal, Vol. 20, No. 4, 2005, pp. 421-424.

doi:10.1002/ffj.1450

[15] L. Castillo, A. González-Coloma, A. González, M. Díaz,

E. Santos, E. Alonso-Paz, M. J. Bassagoda and C. Rossini,

“Screening of Uruguayan Plants for Deterrent Activity

against Insects,” Industrial Crops and Products, Vol. 29,

No. 1, 2009, pp. 235-240.

doi:10.1016/j.indcrop.2008.05.004

[16] INIA, “Estudios en Domesticación y Cultivo de Especies

Medicinales y Aromáticas Nativas. Serie FPTA-INIA

11,” Instituto Nacional de Investigación Agropecuaria

Uruguay, Montevideo, 2004.

[17] S. Echeverrigaray, R. P. Carrer and L. B. Andrade, “Mi-

cropropagation of Salvia guaranitica Benth. through Axil-

lary Shoot Proliferation,” Brazilian Archives of Biology

and Technology, Vol. 53, No. 4, 2010, pp. 883-888.

doi:10.1590/S1516-89132010000400018

[18] E. Haene and G. Aparicio, “100 Árboles Argentinos,”

Albatros, Buenos Aires, 2001.

[19] Administración de Parques Nacionales, “Sistema de In-

formación de Biodiversidad,” Administración de Parques

Nacionales, 2013. http://www.sib.gov.ar/

[20] Sistema Provincial de Áreas Naturales Protegidas, “Lis-

tado de Árboles y Arbustos de Entre Ríos y Especies Ve-

getales en Peligro de Extinción de la Provincia,” Provin-

cia de Entre Ríos, Entre Ríos, 1993.

[21] A. W. Cabrera, “Fitogeografía. Vol. 3,” In: F. de Aparicio

and H. A. Difrieri, Eds., La Argentina. Suma de Geo-

grafía, Peuser, Buenos Aires, 1958, pp. 101-207.

[22] A. L. Cabrera, “Regiones Fitogeográficas Argentinas,” In:

W. F. Kugler, Ed., Enciclopedia Argentina de Agricultura

y Jardinería, Vol. 2, ACME, Buenos Aires, 1994, pp. 1-

85.

[23] M. S. Di Bitetti, G. Placci and L. A. Dietz, “Una Visión

de Biodiversidad para la Ecorregión del Bosque Atlántico

del Alto Paraná: Diseño de un Paisaje para la Conser-

vación de la Biodiversidad y Prioridades para las Ac-

ciones de Conservación,” World Wildlife Fund, Wash-

ington DC, 2003.

[24] M. A. Altieri, “Agroecology: The Science of Sustainable

Agriculture,” Westview Press, Colorado, 1995.

[25] G. N. Agrios, “Plant Pathology,” Elsevier Academic Press,

New York, 2005.

[26] H. M. Alexander, “Disease in Natural Plant Populations,

Communities, and Ecosystems: Insights into Ecological

and Evolutionary Processes,” Plant Disease, Vol. 94, No.

5, 2010, pp. 492-503. doi:10.1094/PDIS-94-5-0492

[27] A. L. Cheheid, E. R. Wright, S. E. Lopez and A. M. R.

Codazzi, “First Report of Alternaria Leaf Spot of Spa-

thiphyllum sp. in Argentina,” Plant Disease, Vol. 84, No.

3, 2000, p. 370. doi:10.1094/PDIS.2000.84.3.370A

[28] E. R. Wright, M. C. Rivera, J. Esperón, A. Cheheid and A.

R. Codazzi, “Alternaria Leaf Spot, Twig Blight, and Fruit

Rot of Highbush Blueberry in Argentina,” Plant Disease,

Vol. 88, No. 12, 2004, p. 1383.

doi:10.1094/PDIS.2004.88.12.1383B

[29] E. G. Simmons, “Alternaria Themes and Variations (73),”

Mycotaxon, Vol. 48, 1993, pp. 109. 140.

[30] P. Neergard, “Danish Species of Alternaria and Stem-

phylium,” Oxford University Press, London, 1945.

[31] E. G. Simmons, “Alternaria: an Identification Manual,”

CBS Fungal Biodiversity Center, Utrercht, 2007.

[32] M. B. Ellis, “Dematiaceous Hyphomycetes,” Common-

wealth Mycological Institute, Kew, 1971.

[33] S. Andrews, “Differentiation of Alternaria Species Iso-

lated from Serials on Dichloran Malt Extract Agar,” In: R.

A. Samson, A. D. Hocking, J. I. Pitt and A. D. King, Ed.,

Modern Methods in Food Mycology, Elsevier, Amster-

dam, 1992, pp. 351-355.

[34] D. F. Farr and A. Y. Rossman, “Fungal Databases,” Sys-

tematic Mycology and Microbiology Laboratory, ARS,

USDA, 2013. http://nt.ars-grin.gov/fungaldatabases/

[35] W. Mulenko, T. Majewski and M. Ruszkiewicz-Michal-

ska, “A Preliminary Checklist of Micromycetes in Po-

land,” Polish Academy of Sciences, W. Szafer Institute of

Botany, Krakow, 2008.

[36] J. Rotem, “The Genus Alternaria, Biology, Epidemiology

and Pathogenicity,” The American Phytopathological So-

ciety, Minnesota, 1994.

[37] J. Lacey, “Water Availability and Fungal Reproduction:

Patterns of Spore Production, Liberation and Dispersal,”

In: P. G. Ayres, L. Boddy, Eds., Water, Fungi and Plants,

Cambridge University Press, Cambridge, 1986, pp. 65-86.

[38] L. W. Timmer, T. L. Peever, Z. V. I. Solel and K. Akimitsu,

“Alternaria Diseases of Citrus—Novel Pathosystems,”

Phytopathologia Mediterranea, Vol. 42, No. 2, 2003, pp.

99-112.

Identification of Alternaria spp. as Pathogenic on the Native Species Terminalia australis and Salvia guaranitica

[39] S. Slavov, S. Mayama and A. Atanassov, “Some Aspects

of Epidemiology of Alternaria alternata Tobacco Patho-

type,” Biotechnology & Biotechnological Equipment, Vol.

18, No. 3, 2004, pp. 85-89.

[40] J. J. Burdon and P. H. Thrall, “Pathogen Evolution across

the Agroecological Interface: Implications for Disease

Management,” Evolutionary Applications, Vol. 1, No. 1,

2008, pp. 57-65. doi:10.1111/j.1752-4571.2007.00005.x