Archives of Phytopathology and Plant Protection
ISSN: 0323-5408 (Print) 1477-2906 (Online) Journal homepage: http://www.tandfonline.com/loi/gapp20
Occurrence of Pythium and Phytopythium species isolated from citrus trees infected with gummosis disease in tunisia Najwa Benfradj, Duccio Migliorini, Nicola Luchi, Alberto Santini & Naima Boughalleb-M’Hamdi To cite this article: Najwa Benfradj, Duccio Migliorini, Nicola Luchi, Alberto Santini & Naima Boughalleb-M’Hamdi (2017) Occurrence of Pythium and Phytopythium species isolated from citrus trees infected with gummosis disease in tunisia, Archives of Phytopathology and Plant Protection, 50:5-6, 286-302, DOI: 10.1080/03235408.2017.1305479 To link to this article: http://dx.doi.org/10.1080/03235408.2017.1305479
Published online: 28 Mar 2017.
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Date: 03 April 2017, At: 07:15
Archives of Phytopathology and Plant Protection, 2017 VOL. 50, NOS. 5–6, 286–302 http://dx.doi.org/10.1080/03235408.2017.1305479
Occurrence of Pythium and Phytopythium species isolated from citrus trees infected with gummosis disease in tunisia Najwa Benfradja , Duccio Migliorinib , Nicola Luchib, Alberto Santinib Naima Boughalleb-M’Hamdia
and
a Département des Sciences Biologiques et de la Protection des Plantes, Institut Supérieur Agronomique de Chott Mariem, Sousse, Tunisie; bInstitute for Sustainable Plant Protection IPSP, National Research Council C.N.R., Sesto Fiorentino, Italy
ABSTRACT
A survey was conducted, during 2012/2013, to identify the causal agents of citrus gummosis in Tunisia. The obtained isolates were identified by molecular and pathological criteria. Three species of Pythium (P. aphanidermatum, P. ultimum and P. dissotocum) and two species of Phytopythium (P. vexans and P. mercuriale) were recovered. Pathogenic tests, using twigs inoculation method for five citrus varieties under laboratory conditions, showed necrotic lesions similar to those observed in natural infected citrus trees by gummosis. In greenhouse experiments, the pathogenicity of the species to citrus was demonstrated by soil and trunk inoculations. Results showed necrotic lesions in the trunk of the variety “Clementine”. P. ultimum is the most virulent, while P. mercuriale was the least virulent in trunk inoculation. Symptom of necrotic roots has been also observed. This is the first report of Pythium and Phytopythium species within the citrus area in Tunisia.
ARTICLE HISTORY
Received 16 May 2016 Accepted 16 February 2017 KEYWORDS
Molecular identification; Oomycota; pathogenicity
Introduction Citrus is an important group of fruit crops world-wide and it ranked third in the world fruit production after apple and banana, and accounts for the production of about 100 million tonnes in a massive area of cultivation spreading over 7.2 million hectares (Savita & Avinash 2012). In Tunisia, the citrus industry is an important production (Khlij & Hajlaoui 2010), covering an area of 34,844 ha and generating about 436,429 tonnes in production in 2013 (FAOSTAT 2015). Recently, field observations reported the appearance of gummosis in all the major citrus growing regions, located in the Northern area of Tunisia (Unpublished data). This disease is one of the most economically important diseases in citrus worldwide (Mounde et al. 2009; Jagtap et al. 2012; Faldoni et al. 2015). Several abiotic CONTACT Naima Boughalleb-M’Hamdi
[email protected]
© 2017 Informa UK Limited, trading as Taylor & Francis Group
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and biotic factors were reported as the main causes of rots and gumming on the trunk and main branches in citrus (Alvarez et al. 2008). Intensive research work on citrus diseases has recently focuses on the role of fungi and fungal-like causing or contributing to the gummosis of citrus, such as Pythiaceae (Erwin & Ribeiro 1996; Sonoda 2000). Phytophthora species have been found as causal agents of gummosis (Verniere et al. 2004) and the most important species are Phytophthora nicotianae and Phytophthora citrophthora (Erwin & Ribeiro 1996; Graham & Menge 2000; Cacciola & Di San Lio 2008). In contrast, little is known about the role played by Pythium species in the development of gummosis of citrus. This genus is abundant in the rhizosphere of diseased citrus trees (Maseko & Coutinho 2002). They are widely distributed throughout the world as soil saprophytes or plant pathogens (Mostowfizadeh-Ghalamfarsa & Banihashemi 2005; Ho 2009) and some of them are important plant pathogens with a worldwide distribution (Matsumoto et al. 1999). Many authors demonstrated that Pythium spp. caused serious reduction in crop yield (Mostowfizadeh-Ghalamfarsa & Banihashemi 2005; Tambong et al. 2006) either as the single pathogen or in complexes with some other fungi. These Peronosporales were reported among the most common fungi from South African citrus orchards (Thompson et al. 1995) and nurseries (Wehner et al. 1986). This combination of Phytophthora and Pythium species may have contributed to increase diseases and represents a major challenge of management strategies. Based on the information stated above, the investigation of Pythium species, in these relatively unexplored habitats, is important to understand not only their ecological roles and physiological features but also the taxonomy and phylogeny of the genus (Uzuhashi et al. 2010). The presence of many species of Pythium makes the identification of disease causal agent difficult because both pathogens and saprobes species can be recovered (Broders et al. 2007). Historically, the identification of different species within the Pythium genus was based on the morphological characters using the keys according to Middleton (1943), Waterhouse (1967) and Plaats-Niterink (1981) and also on growth characteristics on specific media. However, this approach requires skill and time, because these characteristics vary under different culture conditions, there is phenotypic variation within each species and some species are morphologically similar one to another (Li et al. 2014). All this has contributed to potential errors in identification, especially for those lacking many years of experience working with this genus (Lévesque & De Cock 2004). Morphology-based taxonomy is increasingly being supplemented by molecular characteristics of a given species (Paul 2003). Identification of Pythium species through molecular methods such as DNA sequence analysis is very helpful and can provided new evidence and rekindled interest to split the genus Pythium (Ho et al. 2012).The role played by Pythium species in citrus disease in Tunisia is unclear and has not yet been investigated. Therefore, the aims of this study were to (i) determine if Pythium and Phytopythium species are associated with gummosis of citrus in Tunisia using morphological and molecular methods and to (ii) study the pathogenicity of these species on citrus.
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Materials and methods Disease surveys and collection of diseased trunk and soil samples
Twenty-five citrus orchards, located in eight areas of citrus production in Tunisia (Menzel bouzalfa, Benikhled, Soliman, Bouargoub, Takelsa, Akouda, Grombalia and Hawaria) were surveyed in 2012 and 2013. Samples were taken, from soil with roots and trunk of the citrus trees showing symptoms of gummosis, according to the irrigation type (submersion or drip). From each orchard, at least five trees were sampled. For submersion irrigation, sampling was carried out according to the per cent of infested trees in each orchard defined as: (Number of infested trees∕Number of trees in the orchard) × 100
In case of low infestation (per cent of infected trees
