A total of 30 Phormidium strains were established from single filaments. Ten strains (CYN103–CYN112) were isolated from the Waimakariri River mat, four (CYN113, CYN115–117) and eight strains (CYN126–133) were isolated from mat 1 and mat 2 from Hutt River, Site 1 respectively, and eight strains (CYN118–125) were isolated from a mat collected at Hutt River, Site 2.
Anatoxin-a and dihydroanatoxin-a were detected in 18 of the 30 samples. One of these strains (CYN112), isolated from the Waimakariri River, also produced homoanatoxin-a (). All of the strains isolated from mat 1 from the Hutt River, Site 1, produced anatoxins. In contrast not all of the isolates from the other mats produced anatoxins; nine out of ten isolated from the Waimakariri River, one out of eight from the Hutt River, Site 2, and six out of eight from mat 2 from the Hutt River, Site 1 did not contain anatoxins ().
Table 1 Results of the anaF PCR and liquid chromatography-mass spectrometry (LC-MS) of 30 strains of Phormidium isolated from the Waimakariri (WR) and Hutt (HR) rivers. -1-1 = Site 1, mat 1, -2 = Site 2, -1-2 = Site 1, mat 2, ND = Not detected, ATX = anatoxin-a, (more ...)
Among the anatoxin producing strains isolated, the concentration of anatoxins produced varied and there was no relationship between the concentration produced and the original source. For example, total anatoxins produced by the isolates from the Waimakariri River varied from 10.06 to 211.83 mg kg−1 and from 4.75 to 115.42 mg kg−1 dry weight for isolates from mat 1, Hutt River, Site 1. Dihydroanatoxin-a concentrations were always higher than anatoxin-a ().
All strains that were found to produce anatoxins via LC-MS also tested positive by PCR for a segment of the anaF
gene (). Gene segments (404 bp) from strains CYN103–107 and 109–112 were sequenced and were identical. Using Megablast, these segments shared 99% sequence homogeneity with an anaF
gene segment from strain CYN53 (JX088093), a P. autumnale
strain isolated from the Rangitaiki River (North Island, New Zealand; [2
]). The next closest sequence similarities were 94% with Oscillatoria
PCC 6506 (FJ477836), and 91% with Anabaena
sp. (JF803645) and A. flos-aquae
Partial 16S rRNA gene sequences (1340 bp) were obtained for all strains. The strains which tested positive for anatoxins (CYN103–107, 109–113, 115–118, 126–128 and 131-133) were identical over this region (). These strains were also identical to CYN53 (JX088083). These segments had a 99% sequence homology with Tychonema sp. K27 (GQ324965), T. bourrellyi CCAP (AB045897) and T. tenue SAG 4.82 (GQ324973). The sequences from all other strains isolated in this study, except CYN108, were identical, and were 17 nucleotides different from the anatoxin positive strains. When submitted to Megablast the closest matches (99%) for these strains were: Phormidium sp. KU003 (AB094351), Microcoleus vaginatus SEV1-KK3, CJI-U2-KK1, PCC 9802 (EF654076, EF654078, AF284803), P. autumnale SAG 35.90, Ant-Ph68, Arct-Ph5 16S, SAG 78.79 (EF654081, DQ493874, DQ493873, EF654084), Tychonema sp. K27 (GQ324965), T. bourrellyi CCAP (AB045897) and T. tenue SAG 4.82 (GQ324973). Strain CYN108 differed from the other anatoxin negative strains by four nucleotides and an insertion of 10 nucleotides. Megablast analysis returned the same results as obtained for the other anatoxin negative strains.
The 16S rRNA gene sequences were used to construct a phylogenetic tree. The anatoxin and non-anatoxin producing strains isolated in this study clustered in a single clade with 100% bootstrap support (). The anatoxin-producing strains clustered most closely with Tychonema species, whilst the non-toxic strains (CYN108, 119–125, 129, 130) formed their own sub-clade ().
Figure 1 Phylogenetic tree based on 16S rRNA gene sequences (1340 bp). The tree was constructed using the neighbour-joining method. Bootstrap values >50% are noted at the nodes.Scale bar = 0.02 substitutions per site. Ar = Arthrospira, Lep = Leptolyngbya (more ...)
All strains shared a similar morphology although there was considerable heterogeneity among cell lengths and widths. Filaments consisted of a single trichome within a firm colourless sheath. Trichomes were light to dark brown, motile, gradually attenuated towards apical cells, commonly forming hormogonia and not constricted at cross-walls. Cells were generally shorter than wide with some cells having densely arranged granules at cross-walls. Apical cells were rounded and occasionally capitate or calyptrate when mature. Cell dimensions among anatoxin producing strains (CYN103–107, 109–113, 115–118, 126–128 and 131–133) were 5.8–12.5 μm wide by 1.2–7.8 μm long. Among the non-anatoxin-producing strains (CYN108, 119–125, 129–130) the cell dimensions were 6.4–10.6 μm wide by 2.4–4.8 μm long.