Paraphysoderma sedebokerense (P. sedebokerense) (Blastocladiomycota) is a facultative pathogenic chytrid that causes irreversible damage to some green microalgae. Specific attacks leading to culture collapse under different conditions have only been described in the lucrative microalga Haematococcus pluvialis (H. pluvialis), while generating biomass for ketocarotenoid astaxanthin production, both indoors and outdoors. In order to manage the infection, parasite propagules (zoospores/amoeboid swarmers), the initiators of the disease, must be studied. Until now, no report on isolated P. sedebokerense propagules has been published. Here, we report on a reproducible method for the stimulation of P. sedebokerense propagule release and their isolation from fungal cultures in synthetic media and infected H. pluvialis cultures, and we further studied their development under different conditions. The isolated propagules featured different spore morphotypes, with coatless spherical spores and amoeboid swarmers being the most dominant in the first pulse of propagule release in both cultures. Inoculating the pure propagules with the host, in both the presence and absence of nitrogen, resulted in epidemic development in both green and red cells; however, in red cells, the epidemic developed more quickly in the presence of nitrogen. Biologically non-active autoclaved host cells were used to distinguish the initial stages of recognition from more progressive stages of the epidemics; on these cells, propagules encysted but did not develop further. These results prove the existence of heat-stable recognition sites on the host and an obligatory signal transduction from the host to support fungal cyst development. The propagule isolation method described herein is a breakthrough that will enable researchers to study the influence of different substances on the propagules, specifically as the initiators of the infection, and thus assist in the management of chytrid diseases. Moreover, it will be useful in studying host-parasite recognition and, therefore, will increase our understanding of the multiple chytrid infections found in nature.

Although many isolation techniques for algaeaxenic cultures of algae are known, successful isolation approaches are species and contaminant-specific. The commercially important alga Haematococcus lacustris has been intensively studied due to its natural production of the high-value carotenoid astaxanthin, yet there are no reports of axenic H. lacustris cultures. In this article, we describe the successful isolation of axenic H. lacustris, originally isolated in non-axenic form from its natural habitat and adopted to be used daily in vitro, in our laboratory. We verify the absence of bacteria and identify the bacterial communities in the non-axenic algal culture. using PCR amplification with selected universal primers for 16S rRNA gene amplification in the gDNA. Amplification of the 16S rRNA gene of the non-axenic culture showed the presence of bacteria with high identity to Massilia, Blastococcus and Deinococcus species. The bacteria which were identified in our strain further expand our knowledge of bacterial communities on algae, and because we established axenic H. lacustris culture, testing their effect on growth or astaxanthin accumulation in controlled co-cultures is now available. The isolation technique described herein can be applied to other green algal species to eliminate bacteria, and the selected primers can be used to verify the axenic nature of other green algal cultures. The resulting axenic culture is required for molecular genetics research and will be very valuable for establishing heterotrophic growth in laboratory or industrial bioreactors.

The blastocladialean fungus Paraphysoderma sedebokerense parasitizes three microalgae species of economic interest: Haematococcus pluvialis, Chromochloris zofingiensis and Scenedesmus dimorphus. For the first time, we characterized the developmental stages of isolated fungal propagules in H. pluvialis co-culture, finding a generation time of 16 h. We established a patho-system to compare the infection in the three different host species for 48 h, with two different setups to quantify parameters of the infection and parameters of the parasite fitness. The prevalence of the parasite in H. pluvialis and C. zofingiensis cultures was 100%, but only 20% in S. dimorphus culture. The infection of S. dimorphus not only reached lower prevalence but was also qualitatively different; the infection developed preferentially on senescent cells and more resting cysts were produced, being consistent with a reservoir host. In addition, we carried out cross infection experiments and the inoculation of a mixed algal culture containing the three microalgae, to determine the susceptibility of the host species and to investigate the preference of P. sedebokerense for these microalgae. The three tested microalgae showed different susceptibility to P. sedebokerense, which correlates with blastoclad’s preference to the host in the following order: H. pluvialis > C. zofingiensis > S. dimorphus.

Several green algae can divide by multiple fission and spontaneously synchronize their cell cycle with the available light regime. The yields that can be obtained from a microalgal culture are directly affected by cell cycle events. Chromochloris zofingiensis is considered as one of the most promising microalgae for biotechnological applications due to its fast growth and the flexible trophic capabilities. It is intensively investigated in the context of bio-commodities production (carotenoids, storage lipids); however, the pattern of cell-cycle events under common cultivation strategies was not yet characterized for C. zofingiensis. In this study, we have employed fluorescence microscopy to characterize the basic cell-cycle dynamics under batch and continuous modes of phototrophic C. zofingiensis cultivation. Staining with SYBR green—applied in DMSO solution—enabled, for the first time, the clear and simple visualization of polynuclear stages in this microalga. Accordingly, we concluded that C. zofingiensis divides by a consecutive pattern of multiple fission, whereby it spontaneously synchronizes growth and cell division according to the available illumination regime. In high-light continuous culture or low-light batch culture, C. zofingiensis cell-cycle was completed within several light-dark (L/D) cycles (14 h/10 h); however, cell divisions were synchronized with the dark periods only in the high-light continuous culture. In both modes of cultivation, daughter cell release was mainly facilitated by division of 8 and 16-polynuclear cells. The results of this study are of both fundamental and applied science significance and are also important for the development of an efficient nuclear transformation system for C. zofingiensis.

The blastocladialean fungus P. sedebokerense is a facultative parasite of economically important microalgae and for this reason it has gained a lot of interest. P. sedebokerense has a complex life cycle which includes vegetative and resting stages. The resting cysts were assumed to play an essential role in survival by resisting drought, but this ability was never tested and the factors that trigger their formation were not evaluated. This study was aimed to induce resting cyst formation and germination in P. sedebokerense. At first, we tested the survival of P. sedebokerense liquid cultures and found that infectivity is retained for less than two months when the cultures were stored on the bench at room temperature. We noticed that dry cultures retained the infectivity for a longer time. We, thus, developed a method, which is based on dehydration and rehydration of the biomass, to produce, maintain, and germinate resting cysts of P. sedebokerense in both saprophytic and parasitic modes of growth. When the dry cultures were rehydrated and incubated at 30 °C, resting cysts asynchronously germinated after 5 h and the “endosporangium” was protruding outside of the cyst. Our method can be used to preserve P. sedebokerense for research purposes with the advantage of no need for expensive equipment.