Miroslav Vosátka

Phosphatase activity of extra-radical arbuscular mycorrhizal hyphae: A review.

Phosphatase activity of arbuscular mycorrhizal (AM) fungi has attracted attention in three fairly distinct domains: intracellular enzymes with defined metabolic functions that have been studied in intraradical hyphae, histochemical staining of alkaline phosphatase as an indicator of fungal activity measured both intra- and extraradically, and extracellular activity related to mineralization of organic P (Po) compounds that may enhance mycorrhizal utilization of an important nutrient pool in soil. This review focuses on the latter subjects with emphasis on extraradical mycelium (ERM), while it draws on selected data from the vast material available concerning phosphatases of other organisms. We conclude that histochemical staining of alkaline phosphatase is a sensitive and suitable method for monitoring the effect of adverse conditions encountered by ERM both as a symbiotically functional entity in soil, and in vitro without modifying interference of soil or other solid substrates. Furthermore, the quantitative importance of extracellular enzymes for P nutrition of AM plants is estimated to be insignificant. This is concluded from the low quantitative contribution extracellular hyphae of AM fungi give to the total phosphatase activity in soil, and from estimations of which processes that may be rate limiting in organic P mineralization. Maximum values for the former is in the order of a few percent. As for the latter, solubilization of Po seems to be far more important than Po hydrolysis for utilization of Po by AM fungi and plants, as both endogenous soil phosphatase activity and phosphatases of other soil organisms are ubiquitous and abundant. Our discussion of mycorrhizal phosphatases supports the view that extracellular phosphatases of roots and micro-organisms are to a large extent released incidentally into soil, and that the source has limited benefit from its activity.

Effectiveness of indigenous and non-indigenous isolates of arbuscular mycorrhizal fungi in soils from degraded ecosystems and man-made habitats

Culturing in soils from degraded ecosystems significantly influenced the effectiveness of indigenous arbuscular mycorrhizal fungi (AMF) isolated from disturbed and undisturbed soils. The AMF isolates from degraded or artificially created habitats (acid rain polluted site, power station fly ash deposits, spoil banks, pyrite deposit), were not, in most cases, more effective than those from undisturbed soils, when grown in symbiosis with maize in the disturbed soils. Significant effects of soil or substrate on plant growth were found, while the influence of the AMF inoculant was much less pronounced. The development of AMF isolates was reduced in soils with more adverse chemical properties irrespective of the isolate origin. The length of extraradical mycelium of AMF and NADH-diaphorase activity of the mycelium were good indicators of negative effects of stress factors in the soil.

Studies on the diversity of arbuscular mycorrhizal fungi and the efficacy of two native isolates in a highly alkaline anthropogenic sediment

A field survey of the arbuscular mycorrhizal status of herbaceous plant species was conducted in a highly alkaline anthropogenic sediment resulting from the disposal of waste from an acetylene and polyvinyl chloride factory. Most plant species found at the site were mycorrhizal and the dominant mycotrophic plant species was Conyza bilbaoana. Fungal species richness was assessed by identification of spores extracted from the sediment and from continuously propagated trap pot cultures. All of the six species of arbuscular mycorrhizal fungi (AMF) found were from the genus Glomus. Glomus intraradices and G. mosseae were found in field-collected sediment samples and also occurred most frequently in trap cultures. To test the symbiotic effectiveness of these two fungi, seedlings of C. bilbaoana were inoculated with either native G. intraradices BEG163 or G. mosseae BEG198 and non-native G. intraradices BEG75 or G. mosseae BEG25 isolates in sterile and non-sterile sediment collected from the study site. All four isolates were able to colonise C. bilbaoana. However, AMF native to the target sediments were generally more effective than the non-native fungi in promoting plant establishment and growth under highly alkaline conditions. The non-native G. intraradices was, however, more effective than the non-native G. mosseae. The results of this study suggest the use of adapted AMF as inoculants for phytorestoration of alkaline anthropogenic-stressed sediments.

Arbuscular mycorrhiza decreases cadmium phytoextraction by transgenic tobacco with inserted metallothionein

The effect of arbuscular mycorrhiza (AM) on the phytoextraction efficiency of transgenic tobacco with increased ability to tolerate and accumulate cadmium (Cd) was tested in a pot experiment. The tobacco plants bearing the yeast metallothionein CUP1 combined with a polyhistidine cluster were compared to non-transgenic tobacco of the same variety at four Cd concentrations in soil, non-inoculated or inoculated with two isolates of the AM fungus Glomus intraradices. Mycorrhizal inoculation improved the growth of both the transgenic and non-transgenic tobacco and decreased Cd concentrations in shoots and root to shoot translocation. Differences were found between the two AM fungal isolates: one isolate supported more efficient phosphorus uptake and plant growth in the soil without Cd addition, while the other isolate alleviated the inhibitory effect of cadmium on plant growth. The resulting effect of inoculation on Cd accumulation was dependent on Cd level in soil and differed between the more Cd tolerant transgenic plants and the less tolerant non-transgenic plants. Mycorrhiza mostly decreased the phytoextraction efficiency of transgenic plants while increased that of non-transgenic plants at Cd levels in soil inhibitory to tobacco growth. Mechanisms of the observed effects of inoculation on growth and Cd uptake are discussed as well as the possible implications of the results for the exploitation of AM in phytoextraction of heavy metals from contaminated soils.

Treatment with culture fractions from Pseudomonas putida modifies the development of Glomus fistulosum mycorrhiza and the response of potato and maize plants to inoculation

Abstract Among different fractions of liquid Pseudomonas putida culture (living bacterial cells, crude cell extract and low molecular – MW 10 000 fractions) the low molecular fraction showed stimulatory effects on the growth of extraradical mycelium of arbuscular-mycorrhizal fungus Glomus fistulosum Skou and Jakobsen and its alkaline phosphatase activity. The effects were consistently observed in two experiments with potato (Solanum tuberosum L.) and maize (Zea mays L.) cultivated in vermiculite–sand mixture. Living cells of P. putida significantly stimulated growth and alkaline phosphatase activity of extraradical hyphae in the maize experiment only. Mycorrhizal colonization of potato roots was increased in the treatments inoculated with living cells of P. putida or with low-molecular fraction of bacterial culture homogenate added. The total leaf area of maize was significantly increased by inoculation with G. fistulosum alone or together with living cells of P. putida as compared to an uninoculated treatment. Shoot fresh weight, shoot/root ratio and plant height were decreased in treatments receiving low-molecular fraction of bacterial cell homogenate. Inoculation of potato plants with AM fungus increased the weight of the biggest tuber and the total weight of tubers per pot and thus the inoculation has potential use for post-vitro transplanted plants for microtuber production. Low molecular fraction of P. putida culture can be added along with mycorrhizal inoculation for enhancement of mycorrhization and activity of extraradical mycelium of plants.

Differences in the effects of three arbuscular mycorrhizal fungal strains on P and Pb accumulation by maize plants

The effect of arbuscular mycorrhizal (AM) fungi on the accumulation and transport of lead was studied in a pot experiment on maize plants grown in anthropogenically-polluted substrate. The plants remained uninoculated or were inoculated with different Glomus intraradices isolates, either indigenous to the polluted substrate used or reference from non-polluted soil. A considerably lower tolerance to the conditions of polluted substrate was observed for the reference isolate that showed significantly lower frequency of root colonisation as well as arbuscule and vesicule abundance. Plants inoculated with the reference isolate also had significantly lower shoot P concentrations than plants inoculated with the isolate from polluted substrate. Nevertheless, inoculation with either indigenous or reference G. intraradices isolate resulted in higher shoot and root biomass and inoculated plants showed lower Pb concentrations in their shoots than uninoculated plants, regardless of differences in root colonisation. Root biomass of maize plants was divided according to AM-induced colouration into brightly yellow segments intensively colonised by AM fungus and non-colonised or only slightly colonised whitish ones. Intensively colonised segments of the isolate from polluted substrate contained significantly higher concentrations of phosphorus and lead than non-colonised ones, which suggest significant participation of fungal structures in element accumulation.

Long-term tracing of Rhizophagus irregularis isolate BEG140 inoculated on Phalaris arundinacea in a coal mine spoil bank, using mitochondrial large subunit rDNA markers

During the last decade, the application of arbuscular mycorrhizal fungi (AMF) as bioenhancers has increased significantly. However, until now, it has been difficult to verify the inoculation success in terms of fungal symbiont establishment in roots of inoculated plants because specific fungal strains could not be detected within colonized roots. Using mitochondrial large subunit ribosomal DNA, we show that Rhizophagus irregularis (formerly known as Glomus intraradices) isolate BEG140 consists of two different haplotypes. We developed nested PCR assays to specifically trace each of the two haplotypes in the roots of Phalaris arundinacea from a field experiment in a spoil bank of a former coal mine, where BEG140 was used as inoculant. We revealed that despite the relatively high diversity of native R. irregularis strains, R. irregularis BEG140 survived and proliferated successfully in the field experiment and was found significantly more often in the inoculated than control plots. This work is the first one to show tracing of an inoculated AMF isolate in the roots of target plants and to verify its survival and propagation in the field. These results will have implications for basic research on the ecology of AMF at the intraspecific level as well as for commercial users of mycorrhizal inoculation.

Influence of inoculation with arbuscular mycorrhizal fungi on the growth and mycorrhizal infection of transplanted onion

Abstract Effects of inoculation with arbuscular mycorrhizal (AM) fungi on the growth and mycorrhizal infection of onion were observed in five field experiments. Onion seedlings pre-inoculated in multiple trays with peat substratum were transplanted after 6 weeks to the field plots containing steamed, fumigated or non-sterilised soil. Positive growth responses were found after inoculation with most fungal endophytes tested. Inoculated AM fungi were generally more effective than indigenous fungi from non-sterilised soil. The plant growth response to inoculation was higher when indigenous fungi in the soil were eliminated by steam sterilisation or fumigation. In one experiment, inoculation was most effective in non-fertilised and non-irrigated treatments, but the mycorrhizal growth response of the plants inoculated by some fungi was significant even when plants were fertilised and irrigated.

Chitin stimulates development and sporulation of arbuscular mycorrhizal fungi

Abstract Chitin added to sand-soil based cultivation substrates stimulated the root colonization, growth of extraradical mycelium and production of spores of arbuscular mycorrhizal fungi (AMF) in three experiments with Allium amppelloprasum , Plantago lanceolata and Lactuca sativa as host plants. Stimulation of AMF sporulation was also observed when autoclaved mycelium of Fusarium oxysporum was used instead of chitin. Increased numbers of actinomycetes in the substrate as a result of chitin treatment were recorded.

Intracellular colonization ofRhododendron andVaccinium roots byCenococcum geophilum, Geomyces pannorum andMeliniomyces variabilis

Fourin vitro experiments were set up to verify the colonization potential of ectomycorrhizal (EcM)Cenococcum geophilumFr. (strain CGE-4), saprotrophicGeomyces pannorum (Link)Sigler & Carmichael (GPA-1) and a frequent root-associated, potentially ericoid mycorrhiza (ErM)-formingMeliniomyces variabilisHambleton & Sigler (MVA-1) in roots ofRhododendron andVaccinium. A typical ErM fungus,Rhizoscyphus ericae (Read)Zhuang & Korf (RER-1), was included for comparison. All fungal strains intracellularly colonized rootedVaccinium microcuttings: GPA-1 occasionally produced hyphal loops similar to ErM, MVA-1 and RER-1 exhibited a typical ErM colonization pattern. CGE-4 hyphae grew vigorously on and around newly formed roots and rarely penetrated turgescent rhizodermal cells forming intracellular loose loops. Rooting ofRhododendron sp. microcuttings was not promoted by any fungal strain except CGE-4, which also promoted the most vigorous growth ofRhododendron ponticum L. seedlings. The widespread EcM fungusC. geophilum has a potential to colonize non-EcM roots and support their development which may influence overall growth of ericaceous plants. As shown forG. pannorum, structures resembling ErM may be formed by fungi that are to date not regarded as ericoid mycorrhizal.