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. 2016 Jun 10;11(6):e0157017.
doi: 10.1371/journal.pone.0157017. eCollection 2016.

Testing an Alternative Method for Estimating the Length of Fungal Hyphae Using Photomicrography and Image Processing

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Free PMC article

Testing an Alternative Method for Estimating the Length of Fungal Hyphae Using Photomicrography and Image Processing

Qinhua Shen et al. PLoS One. .
Free PMC article

Abstract

This study aimed to develop and test an unbiased and rapid methodology to estimate the length of external arbuscular mycorrhizal fungal (AMF) hyphae in soil. The traditional visual gridline intersection (VGI) method, which consists in a direct visual examination of the intersections of hyphae with gridlines on a microscope eyepiece after aqueous extraction, membrane-filtration, and staining (e.g., with trypan blue), was refined. For this, (i) images of the stained hyphae were taken by using a digital photomicrography technique to avoid the use of the microscope and the method was referred to as "digital gridline intersection" (DGI) method; and (ii), the images taken in (i) were processed and the hyphal length was measured by using ImageJ software, referred to as the "photomicrography-ImageJ processing" (PIP) method. The DGI and PIP methods were tested using known grade lengths of possum fur. Then they were applied to measure the hyphal lengths in soils with contrasting phosphorus (P) fertility status. Linear regressions were obtained between the known lengths (Lknown) of possum fur and the values determined by using either the DGI (LDGI) (LDGI = 0.37 + 0.97 × Lknown, r2 = 0.86) or PIP (LPIP) methods (LPIP = 0.33 + 1.01 × Lknown, r2 = 0.98). There were no significant (P > 0.05) differences between the LDGI and LPIP values. While both methods provided accurate estimation (slope of regression being 1.0), the PIP method was more precise, as reflected by a higher value of r2 and lower coefficients of variation. The average hyphal lengths (6.5-19.4 m g-1) obtained by the use of these methods were in the range of those typically reported in the literature (3-30 m g-1). Roots growing in P-deficient soil developed 2.5 times as many hyphae as roots growing in P-rich soil (17.4 vs 7.2 m g-1). These tests confirmed that the use of digital photomicrography in conjunction with either the grid-line intersection principle or image processing is a suitable method for the measurement of AMF hyphal lengths in soils for comparative investigations.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
The measurement of possum fur on an image taken under a microscopy at ×200 magnification by using (a) the DGI method–a grid layer (12 × 9, grid size 0.05 × 0.05 mm) was placed on the top of the image and the horizontal and vertical intersections of possum fur that crossed the edges of each square were counted and recorded (e.g., C = 14, the possum fur length calculated using the Tenant equation was 0.550 mm), and (b) the PIP method–the possum fur in the same image was traced (yellow line) manually and measured by the ImageJ software (e.g., L = 0.503 mm).
Fig 2
Fig 2
AMF hyphae on an image taken under a microscopy at ×100 magnification measured by using (a) the DGI method—a grid layer (12 × 9, grid size 0.05 × 0.05 mm) was placed on the image, and the horizontal and vertical intersections of hyphae that crossed the edges of each square were counted and recorded (e.g., C = 23, the hyphal length calculated using the Tenant equation was 0.904 mm); and (b) the PIP method—the hyphae in the same image were traced (pink line) and measured by the ImageJ software with NeuronJ plugin (e.g., L = 0.931 mm).
Fig 3
Fig 3. A scatterplot matrix with linear regressions among the lengths of possum fur measured by both the DGI (LDGI) and PIP (LPIP) methods and the known lengths of possum fur (Lknown).
Fig 4
Fig 4. The distribution of the 95% confidence limits (dashed lines) of the mean lengths (solid lines) of possum fur measured by both the DGI (LDGI) and PIP (LPIP) methods and the known lengths of possum fur (Lknown).
Fig 5
Fig 5. The lengths (means ± 95% confidence intervals) of hyphae in soils with low P fertility (solid circles) and high P fertility (open circles) measured by the digital gridline–intersection (DGI) method plotted against measurements by the photomicrography–ImageJ processing (PIP) method.
The 1:1 line is shown as a dashed line.
Fig 6
Fig 6. Ilustration of the intersection of gridlines with a stained hypha and the different number of counts that can be obtained with different random arrangements of the structure.
A given length of 0.35 mm hypha if distributed as (a) intersection count recorded C = 7, then a estimated legnth of 0.28 mm was calculated using Tennant equation, and as (b) intersection count as C = 11, its corresponding estimated length was 0.43 mm.

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Grant support

The New Zealand Biochar Research Centre granted to QHS for this research project at Massey University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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