The current status of Pythium species in Iran: challenges in taxonomy

INTRODUCTION

The genus Pythium Pringsheim 1858 includes various species of fungus-like Oomycota which are living as saprophytes or parasites in soil or fresh water niches. Some of the species, however, are among the well-proven biological control agents of plant pathogenic fungi. Pythium species are pathogenic to many plants and some aquatic and terrestrial animals and they also can parasitize some algae and fungi.

Species of Pythium are ecologically scattered all over the world and considered as cosmopolitan taxa. They can be found in tropical, temperate and even in cold regions. Tropical species can also be recovered from greenhouses in temperate regions (van der Plaats-Niterink 1981). Many of the plant pathogenic Pythium species are economically significant and cause devastating diseases on crops and ornamental plants. Therefore, in order to have a better understanding of biology, ecology and evolutionary relationships among species, identification and characterization of the species are of importance.

The earliest recorded report of a Pythium sp. inducing a plant disease in Iran returns to around 1947, when Esfandiari (1947) reported Pythium debaryanumR. Hesseas the causal agent of tobacco root rot based on his observations. Nevertheless, the first report based on laboratory tests has not been published until 21 years later about Pythium ultimum Trow on lentil (Kaiser et al. 1968) and a few years after, Fatemi (1971) published the first formal description of a Pythium sp. for Pythium aphanidermatum (Edson) Fitzp. from Iran.There has been ongoing attention to the species of this genus ever since. The objectives of this paper are to describe the current taxonomic status of Pythium species in Iran and to illustrate the obstacles existing in the way of identification and description of these species. The paper also reviews the prospects of future studies on taxonomy of the Pythium species in Iran and discusses the solutions for the taxonomic challenges ahead.

Pythium Genus, Species and Groups

There are almost 307 described Pythium species (www.mycobank.org) which are classified in the Kingdom Straminopila (Webster & Weber 2007) and the Phylum Oomycota. The genus concept of Pythium has been an ongoing controversy since the time of the description and there is still no consensus on the alternative genera (e.g. Fischer 1892; Schröter 1897; Sparrow 1931; Waterhouse 1967; van der Plaats-Niterink 1981; Dick 1990; Bala et al. 2010; Ko et al. 2010; Uzuhashiet al. 2010). Most of the authors tried to attend Pythium spp. into other genera based on the sporangial shapes, which is reviewed by Ho (2013). This state of flux forced me to adopt the classical definition of Pythium (Waterhouse 1974), i.e. all zoospore forming oomycetes which produce variable shapes of non-deciduous sporangia in water and in which zoospores form in a membranous vesicle connected to a discharge tube at the tip of the sporangium.

Pythium species are sexually homothallic (self-fertile) or need mating types in heterothallic species to produce oospores. There are some isolates which never produce some of the sexual organs. Theses isolates are categorized into some “Groups” (van der Plaats-Niterink 1981). Some isolates only form sporangia or hyphal swellings, but never produce oogonia or any other sexual organs in single or dual cultures. If these isolates only produce filamentous sporangia, they are grouped as Group “F”. Isolates with swollen, toruloid sporangia are categorized as Group “T”. If these isolates produce globose, non-proliferating sporangia, they are grouped as Group “G”. Globose, proliferating sporangia producing isolates are Group “P”, and isolates which only have hyphal swellings are categorized as Group “HS”.

Taxonomic Criteria for Species Identification

The available identification keys of Pythium species are generally based on morphological, biological and morphometric characteristics (e.g. Matthews 1931; Middleton 1943; Waterhouse 1967; van der Plaats-Niterink 1981; Dick 1990). Therefore, the recognition of these criteria and the factors influencing them will be helpful for accurate identification of the species. The main characteristics for conventional identification of Pythium species are: possessing or lacking the sexual organs (the shapes, sizes and positions of the oogonia the oogonial ornamentations and their sizes; the shapes, sizes, numbers, origins and positions of the antheridia; the sizes, numbers and configurations of the oospores (i.e. if they are plerotic or aplerotic); and the thickness of the oospore wall), as well as asexual organs (the shapes and diameters of the hyphae; the presence and absence of hyphal swellings and their configurations; the presence and absence of chlamydospores, their sizes and colors; the presence and absence of appressoria, their shapes and configurations; the shapes and prolife- rations of zoosporangia), the colony patterns on various media and the temperature relationships.  Due to the simple morphology of the species, the accurate identification of the species requires careful and precise examination of the isolates.

The Taxonomic Challenges of Pythium

Working with Pythium spp. isolates, one might encounter some obstacles during the course of species identification. Some of these complications were related to the biology of these microorganisms and some of which had something to do with the scientific tools and software availability. Most of the Pythium spp. isolates need sterols to produce sexual or asexual reproduction organs, such as zoosporangia and oospores in artificial media. It is possible to add some phytosterols in the form of sterol-rich plant materials, such as hemp seed extract or pure sterols, for instance β-Sitosterol, into media. However, for some isolates it takes a long time to produce any sexual or asexual organs and some of them never produce anything but a mycelial mat of coenocytic hyphae. Additionally, some sexual organs, such as antheridia may decline quickly soon after they appear. This makes the culture observation a time-consuming and laborious task. Some Pythium species produce more than one type of sporangia or antheridia. To avoid any confusion related to this multimorphism, the cultures must be absolutely pure. On the other hand, there is a high level of morphological overlapping among convergent species, especially phylogenetically related ones, which makes them an identification challenge. It is not that easy to find a compatible mating type for a heterothallic Pythium isolate to stimulate oospore production. Therefore, the identification must be solely based on asexual morphology, which is prone to error.

The biology of Pythium species is not the only issue in the course of identification and there are several software-based concerns which have something to do with the accessible tools for the identification. The most comprehensive Pythium species identification key available (van der Plaats-Niternk 1981) only covers 120 out of 307 reported species. Additionally, the latest identification key (Dick 1990) is almost 25 years old. On the other hand, there are also no descriptive sheets, no web-based database and no molecular barcode metadatabase for Pythium species. If a molecular identification is the approach of choice, species-specific primers are designed for only around 20 species (http://sppadbase.ipp.cnr.it/), most of which are developed for plant pathogens. There were some attempts for generating web-based interactive keys based on Lucid Builder platform (Moorman et al. 2014). Nevertheless, it is a modification of van der Plaats-Niternk (1981) identification key and not all the species in the original key are included.

The Current Status of Pythium spp. Identification in Iran

From the timewhen the first Pythium species was formally reported from Iran (Kaiser et al. 1968), 44 species, two varieties, and five groups of this genus have been reported (Table 1) from 76 plant taxa, including 55 plant species, as well as agricultural soil (data not shown). At the moment, there are 371 records of Pythium spp. incidence from various provinces of Iran, which are not evenly distributed in each province (Fig. 1). These reports, however, vary enormously in content, quality, detail, and format. Apart from Fars, Razavi Khorasan, Hamadan, Khuzestan and a few other provinces, there are few or no (e.g. Bushehr, South Khorasan, and Qom) reports in the most provinces.

Table 1. The list of Pythium spp. reported from Iran.

Table 1. Continued

Table 1. Continued

? = unknown.

^† Matrix(ces) and location from which the taxon was reported for the first time in Iran.

The Taxonomic Challenges of Pythium spp. In Iran

In the course of identification and classification of Pythium taxa in Iran, in addition to general obstacles, there are some other problems in the way. There are almost no identification keys for the species from

 Iran. The only available key (Mostowfizadeh-Ghalamfarsa & Banihashemi 2005) just covers the isolates from Fars Province in southern Iran. Furthermore, the shortage of comprehensive checklists of the species, their dispersal and isolation matrices make the confirmation of species identification difficult for the researchers.

Fig. 1. The number of Pythium spp. reports from each province of Iran.