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.

Molecular identification and antibacterial properties of an ericoid associated mycorrhizal fungus.

BACKGROUND: The quest for novel sources of antibacterial compounds have necessitated the inclusion of ericoid mycorrhizal fungi (ERM) commonly found within the root of ericaceous plants. Agar-well diffusion method was used to detect antibacterial activity and was followed by the microbroth diffusion method [minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)]. RESULTS: The results of the phytochemical screening indicated that only alkaloids, flavonoids, phenols, saponins, cardiac glycosides and terpenoids were present, while steroids and tannins were absent. The MIC of the extracts ranged between 2 and 16 mg/mL, and the lowest MIC was obtained with Staphylococcus aureus. Also, the result of the MBC study indicated that the fungal extract was most active at concentrations of 2 and 4 mg/mL against Bacillus subtilis and S. aureus, respectively. CONCLUSIONS: This bioassay showed, for the first time, antibacterial activity of L. incrustata against some bacterial species. Subsequently, ERM fungi should be given attention when searching for antimicrobial agents because they could provide a solution to solve problems associated with conventional disease treatments (i.e. pathogenic microorganisms resistance).

Purification and characterization of an amyloglucosidase from an ericoid mycorrhizal fungus (Leohumicola incrustata).

This study aimed to purify and characterize amyloglucosidase (AMG) from Leohumicola incrustata. AMG was purified to homogeneity from cell-free culture filtrate of an ERM fungus grown in a modified Melin-Norkrans liquid medium. The molecular mass of the AMG was estimated to be 101 kDa by combining the results of Sephadex G-100 gel filtration, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and zymography. The Km and kcat values were 0.38 mg mL-1 and 70 s-1, respectively, using soluble starch as a substrate. The enzyme was stable at 45 °C (pH 5.0), retaining over 65% activity after a pre-incubation period of 24 h. The metal inhibition profile of the AMG showed that Mn2+ and Ca2+ enhanced activity, while it was stable to metals ions, except a few (Al3+, Co2+, Hg2+ and Cd2+) that were inhibitory at a concentration higher than 5 mM. Thin layer chromatography revealed that only glucose was produced as the product of starch hydrolysis. The amylase from L. incrustata is a glucoamylase with promising characteristics such as temperature stability over an extended period, high substrate affinity and stability to a range of chemicals. Also, this study reports for the first time the possibility of using some culturable ERM fungi to produce enzymes for the bio-economy.

Improved endoglucanase production and mycelial biomass of some ericoid fungi.

Fungal species associated with ericaceous plant roots produce a number of enzymes and other bio-active metabolites in order to enhance survival of their host plants in natural environments. This study focussed on endoglucanase production from root associated ericoid mycorrhizal and dark septate endophytic fungal isolates. Out of the five fungal isolates screened, Leohumicola sp. (ChemRU330/PPRI 13195) had the highest relative enzyme activity and was tested along with isolates belonging to Hyloscyphaceae (EdRU083/PPRI 17284) and Leotiomycetes (EdRU002/PPRI 17261) for endoglucanase production under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28 °C. An optimal of pH 5.0 produced enzyme activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and Leohumicola sp. respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone. While NaFe-EDTA and Co2+ inhibited enzyme activity. The potential role of these fungi as a source of novel enzymes is an ongoing objective of this study.

Susceptibility of adult Aethina tumida (Coleoptera: Nitidulidae) to entomopathogenic fungi.

Aethina tumida Murray (Coleoptera: Nitidulidae) is an invasive parasite species in populations of honey bees, Apis mellifera L. Aiming toward substitution of chemical control, we here identified a naturally occurring fungal pathogen of adult A. tumida from its endemic range in South Africa [Metarhizium anisopliae (Metschnikoff) Sorokin variety anisopliae strain FI-203]. The susceptibility of adult beetles (n = 400) to this fungus and to three other generalist entomopathogenic fungal isolates [Metarhizium anisopliae, Beauveria bassiana (Balsamo) Vuillemin, and Hirsutella illustris Minter & Brady] was assessed using spore suspension bioassays. The data revealed significantly increased mortality in the B. bassiana (74.00 +/- 8.94%) and M. anisopliae variety anisopliae (28.00 +/- 16.43%) tests but not in the H. illustris (2.00 +/- 4.47%) and M. anisopliae (12.00 +/- 8.37%) groups. The results indicate a potential for entomopathogenic fungi as an alternative control of A. tumida.