Pythium carbonicum (F-72) sp. nov. was found in soil samples taken on the top of a spoil heap in northern France. The morphology of this new species resembles that of a recently described species: Pythium megacarpum. However, the antheridial and oogonial characteristics of this new species are unique, and the comparison of its ITS region of the nuclear ribosomal DNA indicates that this species is also related to the genus Phytophthora. The fungus does not sporulate, the sporangia germinate directly into mycelium through germ tubes. The oogonia of P. carbonicum are smooth-walled and also papillated, and are provided with monoclinous and diclinous antheridia that wrap around, forming a complicated knot. Morphological features of this new species, together with the sequences of the ITS region of its nuclear ribosomal DNA and its comparison with related species are discussed here.
Pythium carbonicum (F-72) was isolated from samples taken from the top of a spoil heap in Henin-Beaumont, in the north of France. In the region “Nord-pas-de-Calais”, there are numerous such spoil heaps that are remnants of the intense coal mining activity which started around 1850 and came to an abrupt halt around the 1970s. In this region, each city has its spoil heaps which provide some elevation to a normally flat landscape. Spoil heaps are almost barren when seen from far away, but as one climbs on the top some sparse vegetation is found. Spoil-heap samples were collected together with some grass debris, and fungi were isolated. It was very interesting to note the presence of some pythiaceous fungi in the spoil-heap samples.
The genus Pythium with more than 200 described species are distributed throughout the world. They belong to the family of Pythiaceae, order Perenosporales, and class Oomycetes. These were historically classified as fungi, but ultrastructural, biochemical, and molecular sequence analyses strongly indicated that they are in a lineage to the monophyletic Kingdom Straminipilia that includes some algal groups. Most of these live as saprophytes in various types of soil and in aquatic environments. However, some are known to be serious plant pathogens. The most common disease caused by the members of this genus is the ‘damping-off of seedlings’ commonly found in the nurseries.
The most widely used monographs on the taxonomy of the genus Pythium are those of Middleton, Waterhouse and Plaats-Niterink. These taxonomic references are based on comparison of morphological characteristics and temperature–growth relationships. In recent years however, the morphological descriptions are being supplemented by molecular characteristics. The ribosomal gene has been widely used for the genetic identification of many organisms because they are comprised both of sequences that were highly conserved during evolution, and highly variable sequences among species and even within species. The ITS1 (internal transcribed spacer), and ITS2 regions of the ribosomal nuclear DNA are non-conserved regions and can be amplified with the PCR method using universal primers ITS1 and ITS4. The sequences of these regions are used to identify different species within the genus.
P. carbonicum (F-72) was isolated from spoil-heap samples in 1994. At that time, the study undertaken by the author was only based on morphological aspects and the temperature–growth relations of different isolates. The antheridial characters, and the slow growth of this isolate, led to its identification as a species belonging to the group of P. megacarpum. Recently, the ITS regions of the rDNA of certain fungi in the author's collection were amplified, and the sequences revealed that the isolate F-72 is a new taxon, quite different to P. megacarpum and other related fungi. Further studies have made it possible to describe the isolate F-72 as a new species. The name P. carbonicum has been adopted to indicate its presence on the coal mine spoil-heaps. Since its original discovery, the fungus has been maintained in the author's personal collection, and even after years of sub-culturing the fungus still produces reproductive structures. The morphological details of the fungus, the sequences of its PCR amplified ITS region of ribosomal nuclear DNA, and its comparison with related species, are given here.
2Materials and methods
P. carbonicum was isolated from coal-dust samples collected from the top of the ‘Ste Heriette’ spoil-heap situated in the city of ‘Henin-Beaumont’ in the north of France. The spoil-heap samples were taken in sterile capped bottles, together with some grass debris and brought to the laboratory. Fungi were isolated by using the usual baiting techniques on hemp-seed halves in water, and on solid media like PCA (potato carrot agar) and PDA (potato dextrose agar) as described earlier [8–10].
2.2DNA isolation and PCR
The fungus was grown in PDB (potato dextrose broth), which is prepared in the same manner as PDA without the addition of agar–agar. The culture conditions, DNA isolation and the PCR of the internal transcribed spacer (ITS) of the ribosomal nuclear DNA were done by using the procedures described earlier [7,9]. Universal primers ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) were used to amplify the entire ITS region of the rDNA and its sequence was realized by Oligo Express (Paris). The ITS sequence of P. carbonicum has been deposited to the GenBank and its ITS1 sequence has been compared with those of related species.
Sporangia globosa, subglobosa, ovoidia intercalaria, interdum terminalia, 18–30 μm diam., zoosporae non observatae. Oogonia, globosa cum levi pariete, aliquando cum parvis papillis, terminalia et intercalaria, 15–42 μm diam, Antheridia monoclinata et interdum complura ramalia antheridialia complectuntur oogonium formantia nodum circum. Oosporae singulae, interdum duas, pleroticae, globosae 20–32 μm diam, paries 3–5 μm crassus. Incrementum radiale quotidianum 5 mm 25°C in agaro Solani tuberosi et Dauci carotae (PCA). Holotypus in herbario Universitatis Burgundiae conservatus (F-72. Distractum a cumulo faccum venientium e carbonis excavatione in parte septentrionali Franciae.
Mycelium hyaline, well branched. Main hyphae up to 7–8 μm wide. Colonies on PCA are submerged and show a narrow chrysanthemal pattern. Average radial growth of the fungus at 25°C on PCA is 5 mm/day. The fungus grows well in water on hemp-seed halves and produces asexual and sexual structures at room temperatures (18–25°C).
Sporangia (Fig. 1A–D) are globose mostly intercalary at times terminal and sub-terminal; measuring 18–30 μm in diameter (av. 25.6 μm). Zoospores were not observed. The sporangia germinate through stout germ tubes (Fig. 1B–D) to give a new colony without passing through the Sporulation phase.
The fungus reproduces sexually by forming sexual structures on solid media as well as in water on hemp-seed halves. Oogonia are both smooth-walled (Fig. 1E, H) and with small finger like papillae (Fig. 1F, G) which are very different from the wall ornamentations of spiny oogonia found within the genus. The oogonia are spherical, mostly intercalary (Fig. 1E, F), rarely terminal (Fig. 1H), measuring 15–42 μm in diameter (av. 26 μm) and are filled with dense, coarsely granulated protoplasm.
Antheridial branches are very slender and are of monoclinous (Fig. 1H) or diclinous origin, one to many per oogonium. These tend to wrap around the oogonia forming a complicated knot (Fig. 2A–D). The antheridial cells are difficult to observe because of the knot. Antheridia are not persistent and soon disappear after fertilization.
Oospores are of plerotic type (Fig. 2E, F), usually one, infrequently two per oogonium (Fig. 2G, H), spherical, measuring 14–32 μm in diameter (av. 25.7 μm). The oospore walls are thick, measuring between 3 and 5 μm (av. 3.5 μm).
The ITS region of the nuclear ribosomal DNA of P. carbonicum (F-72) has the GenBank accession number, AY191003, and is as following:
The comparison of the ITS1 sequences of P. carbonicum and related species is given in Fig. 3 in the form of CLUSTAL multiple alignment.
P. carbonicum is unique because no other species in the genus has the character combination of slow growth, absence of zoospores, antheridia wrapping around the oogonia, presence of papillated oogonia, and thick-walled oospores. Morphologically this species is close to a recently described species, Pythium megacarpum, as both species possess thick-walled oospores and antheridia wrapping around the oogonia. However, the antheridial cells are large and quite conspicuous with oospore walls up to 7 μm in thickness in the case of P. megacarpum, while the antheridial cells are inconspicuous with oospore walls only up to 5 μm thick in the case of P. carbonicum. The temperature–growth relationship of both these species is also different. The new species possess, at times, papillated oogonial walls. Only one species, P. ornacarpum, has the character combination of coiled and wrapping type of antheridia and oogonial projections. However this species is quite different from P. carbonicum as the oogonia of the former has real pointed spines and not just papillae of the latter.
A blast query with the ITS sequence of P. carbonicum gave the closest score of 78.2% similarities with an undescribed species of Pythium isolated from the Bavarian alps (accession AY162278), Pythium sp. 334. Other scores are: 64.4% with Pythium oedochilum (AF244124), 63.2% with P. megacarpum (AF242896), 61.7% with P. vexans (AF452137), 60.4% with P. ornacarpum (AF187840), and 70.5% with Phytophthora cinnamomi (L76535). Hence the new species is also very close to the genus Phytophthora suggesting its position as a link between the two genera. However, the ITS sequence of P. carbonicum is nowhere near that of any described species of either Pythium or Phytophthora.
A comparison of the ITS regions of these fungi can be observed in the multiple alignment in Fig. 3. The morphological differences between P. carbonicum and other related species, as well as those in the sequences of the ITS region, justifies the creation of this new taxon.