This analysis is a NLFA derived analysis and based on the work by Sharma and Buyer (2015). “Comparison of biochemical and microscopic methods for quantification of arbuscular mycorrhizal fungi in soils and roots.”
The soil spore density is correlated to the NLFA measurement at the biomarker position 16:1w5 which is the VAM biomarker. The NLFA is a better analysis for specifically VAM as this biomarker on a PLFA is highly compromised with certain bacteria and does not provide an accurate measure for Mycorrhizae.
This test is also based on a correlation curve established with microscopy root colonization percentage data correlated with NLFA (Neutral Lipid Fatty Acid) analysis expressed in ng/g at the biomarker 16:1w5 where a 0.74 correlation coefficient was achieved.
Evaluating Their Conclusion:
- NLFA as an Indicator of Root Colonization
- Suppose Sharma and Buyer found higher NLFA levels in colonized roots. In that case, it suggests that mycorrhizal fungi store lipids within intraradical structures (arbuscules, vesicles, hyphae inside roots) rather than mainly in spores.
- This aligns with studies showing that AM fungi store energy as neutral lipids (triglycerides) within root-colonizing structures, meaning NLFA may directly reflect the fungal presence inside roots rather than just dormant spores.
- PLFA vs. NLFA in Mycorrhizal Detection
- PLFA (membrane lipids) degrades quickly after cell death, making it a reliable marker for active hyphal biomass in the rhizosphere.
- NLFA (storage lipids) is more stable and accumulates in intracellular fungal structures within roots, possibly making it a more sensitive long-term indicator of root colonization.
- Some research suggests that NLFA 16:1ω5 increases as AM fungi develop vesicles inside root cells, further supporting its relevance for root colonization.
- Possible Explanation for Sharma & Buyer’s Findings
- If NLFA correlates more strongly with root colonization than PLFA, it may be because the majority of the fungal biomass inside roots is involved in storage rather than active membrane expansion.
- PLFA 16:1ω5 might better reflect external hyphal networks, whereas NLFA 16:1ω5 could better measure fungal presence within roots.
- If the goal is to measure external mycorrhizal activity and nutrient exchange → PLFA 16:1ω5 is likely better.
- If the goal is to measure overall root colonization, including fungal storage within roots → NLFA 16:1ω5 might be more accurate.
Thus, while NLFA appears to be a strong indicator of root colonization, it is best used in combination with PLFA for a more comprehensive assessment of mycorrhizal activity in roots and surrounding soil.
How to take the root samples for PLFA and NLFA analyses for evaluation of VAM root colonization percentage.
The best time for corn is between the V4 and V7 stage.
- Dig up the plant keeping the roots intact.
- Wash the soil off the roots carefully to cause as little damage as possible to the fine hair roots.
- Cut the stem off.
- Separate the different main root branches from each other and select the youngest fibrous roots at the bottom end.
- Cut these young fibrous roots off from the main root branch as shown below.
- Wash these roots very carefully so that all the soil is removed, Sand the roots have a clean white appearance.
- Select the bottom portion of these roots that contain the youngest root hairs for the sample which should look like the pic below.
- This also gives an idea of the root volume required.
- Place the final selected root samples between two paper towels and place the samples wrapped between the paper towels in a zip lock bag and label appropriately.
Freeze the samples as soon as possible and ship in an insulated container with ice bags.
