Pythium longandrum (F-73.0) was isolated, from soil samples taken in Lille in northern France. Morphologically the fungus resembles closely Pythium rostratum, however its antheridial characters are unique. The oogonia of this species are provided with hypogynous and monoclinous antheridia. The antheridial cells are inflated and are probably the largest and longest for the genus. The internal transcribed spacer region of its nuclear ribosomal DNA indicates that it is entirely different from all other species of Pythium. This new species is characterized by its spherical to elongated sporangia, smooth-walled oogonia and hypogynous to monoclinous antheridia bearing long antheridial cells closely applied to the oogonia. Morphological features of this new species, together with the sequences of the ITS region of its nuclear ribosomal DNA and comparison with related species are discussed here.
The members of the genus Pythium are well known and are represented worldwide. These fungi belong to the family of Pythiaceae, order Perenosporales, and class Oomycetes. More than 200 species of this genus have been described out of which 130 have been recognized recently . Most live as saprophytes in various types of soil and in aquatic environments, some however can become 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. Some can also behave as parasites of mosquito larvae  and one is a well known mammalian parasite .
The taxonomic description of the genus Pythium has been dealt with from time to time, but the most widely used monographs are those by Middleton , Waterhouse  and Plaats-Niterink . These taxonomic references are based on the comparison of morphological characteristics and temperature–growth relationships of different members of the genus. In recent days the morphological descriptions are increasingly supplemented by molecular characteristics of a given species . The polymerase chain reaction coupled to restricted fragment length polymorphism analysis (PCR-RFLP) has become a useful tool in fungal taxonomy [8,9] and is currently used to identify different species of Pythium[10–13]. Amplification of the ribosomal gene is used for the genetic identification of many organisms because they comprise both highly conserved sequences during evolution and highly variable sequences among species and even within species. The ribosomal nuclear DNA consists of transcribed and non-transcribed regions. The ITS1 (internal transcribed spacer 1), and ITS2 are non-conserved regions and can be amplified with the PCR method using universal primers ITS1 and ITS4 .
During the course of investigation on pythiaceous fungi, Pythium longandrum (F-73.0) was isolated from soil samples taken in a wheat field in the outskirts of Lille in northern France in 1991. The study on the fungus was then purely morphological and despite some differences regarding antheridial characters, it was mistakenly identified as the very commonly found, Pythium rostratum and kept aside for 10 years. Recently the ITS regions of the rRNA of certain fungi in the author's collection were amplified and the sequences reveal that the isolate F-73.0 was wrongly identified as P. rostratum. Further studies have made it possible to describe the isolate F-73.0 as a new species. The name P. longandrum has been adopted due to the presence of very long and wide antheridial cells, perhaps the longest for the genus. Since its original discovery, the fungus is being maintained in the author's personal collection and even after years of sub-culturing the fungus still produces plenty asexual and sexual structures. The morphological and reproductive details of this fungus, the sequences of the PCR-amplified ITS region of ribosomal nuclear DNA, and its comparison with related species, are discussed in this article.
2Materials and methods
All fungal isolates of Pythium were taken from the author's personal collection of fungi and were grown on hemp-seed halves in water, and on solid media like PCA (potato carrot agar) and PDA (potato dextrose agar) as described earlier [8,15,16].
2.2DNA isolation and PCR
The fungi were 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 ITS1 of the ribosomal nuclear DNA was done using the procedures described earlier [15,17]. Universal primers ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) were synthesized and the DNA sequence was realized by Oligo Express (Paris, France). ITS1 is at the 3′ end of the 18S rDNA gene and ITS4 is at the 5′ end of the 28S rDNA gene. The sequences obtained were compared with the ITS sequences of related species of Pythium: P. rostratum, P. orthogonon, P. middletonii, P. ultimum, P. undulatum, and P. terrestris (F-78, unpublished). The sequence of the ITS region of the nuclear ribosomal DNA of P. longandrum has been deposited in GenBank.
Sporangia globosa, subglobosa, intercalaria, interdum terminalia, 16–36 μm diameter, zoosporae non-observata. Oogonia laevia, globosa, interdum ovoidia, terminalia et intercalaria, 18–26 μm diameter, Antheridia hypogynata vel monoclinata, cellulae antheridiales inflatae. Oosporae singulae, apleroticae et pleroticae, globosae 17–23 μm diameter, paries 1.5–2 μm crassus. Incrementum radiale quotiadianum 7–8 mm 25°C in agaro Solani tuberosi et Dauci carotae (PCA).
Etymology: The fungus is being named as P. longandrum because of the presence of long and inflated antheridial cells.
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 7 mm/day. The fungus grows well in water on hemp-seed halves and produces asexual and sexual structures at room temperature (18–25°C).
Sporangia (Fig. 1a–c) are globose to somewhat elongated, mostly intercalary and catenulate, but at times terminal and subterminal; measuring 16–36 μm ID diameter (average 29.4 μm). Zoospores were not observed, but on the other hand long discharge tubes were observed which indicates that the sporangia can germinate directly to give a new mycelium without passing through the sporulation phase.
The fungus reproduces sexually by forming antheridia and oogonia (Fig. 1d–j) on solid media as well as in water on hemp-seed halves in abundance. Oogonia are smooth-walled, spherical, at times elongated, terminal, subterminal or intercalary, measuring 17–26 μm in diameter (average 21.5 μm) and are filled with dense, coarsely granulated protoplasm.
Antheridia are of hypogynous (Fig. 1d–g) and also of monoclinous origin (Fig. 1h–j). The antheridial cells are very peculiar. These are very much inflated, at times bilobed into two conspicuous cells (Fig. 1d,f,g). At times the antheridial cells are very long and longitudinally applied to the oogonia (Fig. 1h–j). The antheridial cells of this species can measure up to 40–50 μm in length and 8–9 μm in breadth. These are probably the longest antheridial cells for the genus. At times the antheridial cells send two or more fertilization tubes into the oogonia (Fig. 1h,i).
Oospores are plerotic (Fig. 2b,c) or aplerotic (Fig. 2a), spherical, usually one but at times two oospores per oogonium (Fig. 2d), measuring 18–23 μm in diameter (average 20.1 μm). The oospore wall is relatively thin, 1–2.5 μm thick.
The ITS region of the nuclear ribosomal DNA of the fungus is comprised of 560 bases (GenBank accession AY039713):
The comparison of the ITS sequences of P. longandrum and related species is given in Fig. 3 in the form of CLUSTAL multiple alignment.
P. longandrum falls in the group of Pythium species that are slow growing and whose oogonia are supplied with mostly hypogynous antheridia. Fungi like P. rostratum, P. hypogynum, P. pulchrum, P. ultimum have this type of antheridia, but none of these have the giant antheridial cells of P. longandrum. A query sent to BLAST regarding the sequences of the ITS region of its nuclear ribosomal DNA, gives a close resemblance with P. orthogonon (accession AJ 233452, 76.7% similarity), P. middletonii (accession AJ 233449, 76.7% similarity), P. ultimum (accession AF 271225, 71.8% similarity), P. rostratum (accession AJ 233456, 64.9% similarity), and P. undulatum (accession AF 271230, 62.3% similarity). The fungus F-78 maintained in the author's collection as P. terrestris (unpublished, accession AY039714) is much closer to P. longandrum with 81.9% similarity. A comparison of the ITS regions of these fungi can be observed in the multiple sequence alignment in Fig. 3. The morphological differences between P. longandrum and other related species, as well as those in the sequences of the ITS region, justifies the creation of this new taxon.