New report of Alternaria alternata causing leaf spot of Aloe vera in Pakistan

Abstract

A leaf spot disease was observed on Aloe vera plants as small, circular to oval dark brown necrotic sunken spots on the leaves. Infected tissues collected from different sites in diseased fields were cultured on malt extract agar medium, and the pathogen was identified as Alternaria alternata on the basis of morphological and cultural characteristics. The fungus produced effuse, olivaceous black colonies with dark olive-green margins, and abundant branched septate, golden brown mycelium. The conidiophores were branched, straight, golden-brown, smooth-walled, measuring up to 60 μm long by 3 μm wide with one conidial scar. The conidia were obpyriform, golden-brown, smooth-walled, produced in long branched chains, with a short pale beak. Pathogenicity tests conducted on healthy potted aloe plants in a glasshouse showed typical leaf spot symptoms after four to seven days. This is the first report of a leaf spot disease of A. vera in Pakistan.

Résumé

Une forme d'alternariose a été observée sur l'Aloe vera. Les symptômes de cette maladie se manifestent par de petites taches creuses nécrotiques, rondes ou ovales, brun foncé, sur les feuilles. Des tissus infectés collectés en différents endroits dans des champs infestés ont été cultivés sur gélose à l'extrait de malt, ce qui a permis, sur la base des caractéristiques morphologiques et culturales, d'identifier l'agent pathogène en tant qu'Alternaria alternata. Le champignon produit des colonies diffuses noir olive dont les marges sont vert olive foncé, possédant de nombreuses cloisons ramifiées et un mycélium brun doré. Les conidiophores étaient ramifiés et droits, brun doré, à paroi lisse, mesurant jusqu'à 60 μm de long sur 3 μm de large, et portant une cicatrice conidiale. Les conidies étaient obpyriformes, brun doré, à paroi lisse, produites en longues chaînes ramifiées et portaient un court bec pâle. Des tests de pathogénicité effectués en serre sur des plants sains d'aloès en pot ont produit les symptômes typiques au bout de quatre à sept jours. Il s'agit du premier rapport d'alternariose chez A. vera au Pakistan.

Keywords:

• Aloe vera
• Alternaria alternata
• leaf spot
• obpyriform conidia

Mots clés:

• Aloe vera,
• Alternaria alternata,
• alternariose
• conidies obpyriformes

Introduction

Aloe vera (L.) Burm f. (syn. Aloe barbadensis Miller) is a tropical plant of hot and dry climates and is widely distributed in Asia, Africa and other tropical regions of the world. It is a perennial herb with fleshy leaves and is grown for its therapeutic and cosmetic use and is also consumed as a vegetable in some parts of the world. There are not many published reports in the literature on diseases of this medicinal herb, perhaps because this plant may be resistant to most fungal and bacterial infections. In fact, some studies have reported antimicrobial properties associated with this plant (de Rodríguez et al., 2005). A few leaf spot diseases caused by Fusarium spp. were reported from the USA (Farr et al., 1989) and Japan (Hirooka et al., 2007), and a leaf spot caused by Alternaria alternata (Fr.) Keissler was reported from India (Kamalakannan et al., 2008). In India, A. alternata is one of the most serious fungal diseases affecting the commercial cultivation of A. vera in North India (Shukla et al., 2008). In previous disease reports, information was absent with regard to plant diseases in Pakistan and particularly Alternaria diseases on medicinal crops such as A. vera (Shakir et al., 1997).

Aloe vera has been cultivated on a commercial scale at the Herbal Heritage Center, Institute of Mycology and Plant Pathology, University of the Punjab, Lahore, Pakistan for the preparation of aloe gel and juice. During July 2009, a leaf spot disease was observed in an A. vera field, and by December, the disease had spread and a significant number of plants were infected. The objective of this study was to describe this new leaf spot disease of A. vera, and to isolate and identify the causal organism and confirm its pathogenicity on healthy potted aloe plants in a glasshouse.

Materials and methods

Sampling and isolation

Eleven infected leaf samples were collected from different parts of aloe fields located at the Herbal Heritage Center, University of the Punjab, and brought to the laboratory for isolations. Leaf spot tissues were cut into small pieces with a sterilized scalpel, surface sterilized with 70% ethanol for 30 s followed by 0.5% NaOCl for 2 min and plated on 2% (w/v) autoclaved malt extract agar (MEA) medium. Plates were incubated at 25 ± 2 °C for six days. Microscopic examinations were conducted by mounting fungal tissues in water and lactophenol, and dimensions of conidia were measured from six-day-old cultures.

Pathogenicity test

Pathogenicity of the isolated organism was confirmed on healthy Aloe vera plants, grown in pots in a glasshouse. Two randomly selected leaves from each plant were pin pricked and spray inoculated with spore suspension (2×10⁴ conidia mL⁻¹) of the pathogen. Leaves sprayed with sterile distilled water served as a control. Typical symptoms were produced on the inoculated leaves after four to seven days. The pathogen from the infected leaves was re-isolated on 2% MEA medium as described above. The morphological and cultural characteristics of the re-isolated organism were compared with the original pathogen. The pathogen was identified from all infected leaf samples.

Results and discussion

A leaf spot disease affected a significant number of plants and caused heavy losses (Fig. 1a ). Small, circular to oval, necrotic and sunken dark brown spots on the leaf, ranging from less than 1.0 mm to an average diameter of 3.0 mm were typical symptoms of disease (Fig. 1b ). Characteristic leaf spots had grey centres with brown margins. In later stages of infection, the affected leaves started yellowing and dried from the tip downwards with loss of leaf texture and mucilaginous jelly (Fig. 1c ). The same pathogen was isolated from all infected samples and no other pathogenic fungus was isolated from the leaf spots. The fungus grew well on 2% MEA, and formed an effuse, olivaceous black colony with dark olive green margins, reverse black and of about 9 cm in diameter at 25 ± 2 °C in six days. The fungus produced abundant branched septate, golden brown mycelium. Conidiophores arose singly, simple, straight, golden brown, smooth up to 60 μm long, 3 μm thick with one conidial scar. Conidia formed in long and branched chains, obpyriform, golden brown, smooth with up to eight transverse septa with three to four longitudinal septa, overall length 20–48 μm, 8–10 μm thick in the broadest part with pale short beak up to 3 μm thick (Fig. 1d ). The isolated fungus was identified as Alternaria alternata (Fr.) Keissler based on symptoms on aloe and morphological and cultural characteristics (Ellis, 1971; Shakir et al., 1997).

Fig. 1. a, Aloe field infected with leaf spot disease caused by Alternaria alternata. b, Leaf spots symptoms on aloe leaves. c, Infected leaves of Aloe vera. d, Conidia of Alternaria alternata at 40×.

According to the literature, this is the first report of a leaf spot disease of A. vera in Pakistan. Pathogenicity tests conducted on healthy potted A. vera plants in a glasshouse produced typical symptoms on leaves after four to seven days. The pathogen was re-isolated and identified from all infected leaf samples. The control leaves did not show any symptoms. Alternaria alternata is a common plant pathogen, which causes a variety of diseases of fruits and plants. Previously, A. alternata has been reported in Pakistan as a pathogen of tomato causing postharvest losses in high frequency (Akhtar et al., 1994) and as a leaf spot pathogen of potato (Solanum tuberosum L.) (Khan & Ahmad, 1992), tomato (Lycopersicon esculentum L.) (Akhtar et al., 2004), lambsquarter (Chenopodium album L.), and toothed dock (Rumex dentatus L.) (Siddiqui et al., 2009a , 2009b ). A culture of A. alternata has also been deposited at the Institute of Mycology and Plant Pathology, University of The Punjab, Lahore, Pakistan, for further studies.