Methods for assessing the quality of AM fungal bio-fertilizer: Retrospect and future directions.

In the recent past, the mass production of arbuscular mycorrhizal (AM) fungi has bloomed into a large biofertilizer industry. Due to their obligate symbiotic nature, these fungi are propagated on living roots in substrate-based pot cultures and RiTDNA in in vitro or root organ culture systems. The quality assessment of AM inocula remains critical for the production and efficacy evaluation of AM fungi. The vigour of AM inocula are assessed through microscopic methods such as inoculum potential, infectivity potential/infection units, most probable number (MPN) and spore density. These methods marginally depend on the researcher's skill. The signature lipids specific to AM fungi, e.g. 16:1ω5cis ester-linked, phospholipid, and neutral lipid fatty acids provide more robustness and reproducibility. The quantitative real-time PCR of AM fungal taxa specific primers and probes analyzing gene copy number is also increasingly used. This article intends to sensitize AM fungal researchers and inoculum manufacturers to various methods of assessing the quality of AM inocula addressing their merits and demerits. This will help AM producers to fulfil the regulatory requirements ensuring the supply of high-quality AM inocula to end-users, and tap a new dimension of AM research in the commercial production of AM fungi and its application in sustainable plant production systems.

Inhibition of a ubiquitously expressed pectin methyl esterase in Solanum tuberosum L. affects plant growth, leaf growth polarity, and ion partitioning.

Two pectin methyl esterases (PMEs; EC 3.1.1.11) from Solanum tuberosum were isolated and their expression characterised. One partial clone ( pest1) was expressed in leaves and fruit tissue, while pest2 was a functional full-length clone and was expressed ubiquitously, with a preference for aerial organs. Potato plants were transformed with a chimeric antisense construct that was designed to simultaneously inhibit pest1 and pest2 transcript accumulation; however, reduction of mRNA levels was confined to pest2. The decrease in pest2 transcript was accompanied by up to 50% inhibition of total PME activity, which was probably due to the reduction of only one PME isoform. PME inhibition affected plant development as reflected by smaller stem elongation rates of selected transformants when compared with control plants, leading to a reduction in height throughout the entire course of development. Expansion rates of young developing leaves were measured simultaneously by two displacement transducers in the direction of the leaf tip (proximal-distal axis) and in the perpendicular direction (medial-lateral axis). Significant differences in leaf growth patterns were detected between wild-type and transgenic plants. We suggest that these visual phenotypes could be correlated with modifications of ion accumulation and partitioning within the transgenic plants. The ion-binding capacities of cell walls from PME-inhibited plants were specifically modified as they preferentially bound more sodium, but less potassium and calcium. X-ray microanalysis also indicated an increase in the concentration of several ions within the leaf apoplast of transgenic plants. Moreover, quantification of the total content of major cations revealed differences specific for a given element between the leaves of PME-inhibited and wild-type plants. Reduced growth rates might also be due to effects of PME inhibition on pectin metabolism, predominantly illustrated by an accumulation of galacturonic acid over other cell wall components.

Uptake and transfer of nutrients in ectomycorrhizal associations: interactions between photosynthesis and phosphate nutrition.

Energy-dispersive X-ray microanalytical investigations and microautoradiographic studies were carried out to examine whether the uptake and transfer of phosphate (P) by an ectomycorrhizal fungus is affected by the carbohydrate supply of its host plant. For this purpose, non-mycorrhizal seedlings of Pinus sylvestris L. and plants inoculated with the ectomycorrhizal basidiomycete Suillus bovinus (L. ex Fr.) Kuntze were placed in the dark for 7 days in advance of a P supply. The subcellular element distribution and the uptake and distribution of (33)P was analyzed in non-mycorrhizal and mycorrhizal roots of these plants and compared with plants kept constantly under normal light conditions (control plants). The results show that placing non-mycorrhizal plants in the dark in advance of the nutrient supply led to (1) a reduction of the subcellular contents of P, S and K, but to an increase in the cytoplasmic Na content, and (2) an increase of (33)P absorption and translocation to the shoot. It can be assumed that this increased inflow of (33)P in non-mycorrhizal plants was due to P starvation after suppressed photosynthesis and reduced respiration of these plants. The suppression of photosynthesis by an ectomycorrhizal host plant and the resulting lower carbohydrate supply conditions for the ectomycorrhizal fungus led to (1) a decrease of P absorption by the mycobiont, (2) a change of the P allocation in the fungal cell compartments of an ectomycorrhizal root, and (3) a reduction of P transfer to the host plant. However, microautoradiographic studies revealed that, under these conditions, P was also absorbed by the mycorrhizal fungus and translocated via the Hartig net to the host plant. In mycorrhizal roots of plants placed in the dark in advance of the nutrient supply, the cytoplasmic P content of the Hartig net was reduced and, instead, a high number of polyphosphate granules could be detected within the hyphae. The results indicate that the exchange processes between the symbionts in a mycorrhiza are possibly linked and that P uptake and translocation by an ectomycorrhizal fungus is also regulated by the carbohydrate supply of its host plant.

Microautoradiographic localization of phosphate and carbohydrates in mycorrhizal roots of Populus tremula x Populus alba and the implications for transfer processes in ectomycorrhizal associations.

Microautoradiographic studies were carried out to examine the distribution and exchange of phosphate and labeled carbohydrates in mycorrhizal roots of Populus tremula x Populus alba L. following application of 33P-orthophosphate (Pi) and 14CO2. Labeled Pi was not homogeneously distributed along the mycorrhizal longitudinal axis. The fungal sheath and the Hartig net contained more 33Pi in the median parts of the root than in the apical or basal root zones, indicating that uptake and transfer of Pi to the host plant was localized mainly in this area. The Pi was translocated by the Hartig net and the interfacial apoplast to the host plant. It was distributed by way of the stele within the plant. Young leaves and meristematic tissue in the shoot tip were the main sinks for Pi. In plants that were left in the dark for 5 days before 33Pi application, the reduced carbohydrate supply caused a decrease in Pi absorption by mycorrhizal roots. Microautoradiography of mycorrhizal roots after assimilation of 14CO2 revealed that: (1) the fungal partner had a high capacity to attract photosynthates; (2) the main transfer of carbohydrates was localized in the median zone of a mycorrhizal root; (3) carbohydrates that were absorbed by the mycorrhizal fungus were translocated to the fungal sheath and were homogeneously distributed; and (4) in the main exchange zone, cortical cell nuclei showed a high sink capacity, indicating increased metabolic activity in these cells. We postulate that (1) the phosphate demand of the host plant regulates absorption of Pi by the fungus, and (2) a bidirectional transfer of carbohydrates and Pi occurs across the same interface structure in ectomycorrhizal roots of Populus.