Quantitative assessment of ectomycorrhiza

The most important measurement in any ectomycorrhizal research study, especially seedling studies, is the quantitative assessment of the ectomycorrhiza. Reliable quantification of ectomycorrhiza is essential to determine inoculum efficacy, ectomycorrhizal contribution to seedling biomass, nutrient absorption, relationships to root pathogen infection, etc. There is no one assessment technique that is, or should be, used for all studies. The choice of technique should be determined by the objectives of the study. There are two general types of assessments: visual estimates and direct counts (Grand and Harvey, 1982). In many seedling studies, entire root systems are examined with the unaided eye or at 5x magnification and the percentages of all short roots with specific ectomycorrhiza are visually estimated. An experienced observer can separate the percentage of those formed by individual fungi to within 5%. After estimation, the numbers can be assigned to rating systems such as + (< 1%), ++ (< 10%), + ++(<30%), and + + + +

(>30%), or excellent (75-100%), good (50-74%), moderate (24-49%), or poor (1-24%). Marx (1981) developed an index for assessing seedlings with Pt ectomycorrhiza. The Pt index is computed using the formula a x b/c where a = percentage of seedlings with any percentage of Pt ectomycorrhizae, b = average percentage of feeder roots with Pt ectomycorrhiza (including those with 0%), and c = average percentage of total ectomycorrhiza formed by all fungi including Pt. The Pt index can range from 0 to 100 and it provides a single value which represents fungus aggressiveness, inoculum efficacy and field performance. Other researchers have used modifications of this index for other fungi (Le Tacon et al, 1988).

Direct counts of entire root systems have been used when only a few seedlings are involved. Each short root and ectomycorrhiza is counted under magnification and lateral root lengths are measured. The data can be presented as total number of ectomycorrhiza per seedling, number of ectomycorrhiza cm-1 of lateral root, or percentage of short roots that are ectomycorrhizal. Ectomycorrhiza on randomly selected lateral roots (cut from the plant) have also been counted directly on larger seedlings. Ectomycorrhiza can also be quantified by counting tips, by computing the average surface area, and by weighing the ectomycorrhiza.

Recently, Marx (1990) developed an assessment procedure involving a photocopying machine to determine lateral root length, number and percentage of ectomycorrhiza, and morphology of ectomycorrhiza. This procedure is as follows. Entire root systems are washed thoroughly of all debris. Short roots are counted on 1-cm segments of 5-10 randomly selected lateral roots still intact on each root system. Percentages of short roots ectomycorrhizal with Pt or other fungi are visually estimated twice at 5x magnification for each entire root system. The two estimates are averaged if they do not vary by more than 10%. If variation is more than 10%, a third estimate is made and the three estimates are averaged.

In order to reflect relative size differences in morphology of the Pt ectomycorrhiza a size estimate between 1 and 10 (Table I) is assigned on the basis of visual estimates of the proportions of ectomycorrhiza by morphological type on each entire root system. These estimates are made twice and averaged for each seedling. This number (between 1 and 10) is multiplied by the number of ectomycorrhiza (determined later) to reflect more accurately the total rating of ectomycorrhiza on each root system. After ectomycorrhizal assessment, first-order lateral roots (all lateral roots attached to the taproot) are removed from each seedling, placed between two sheets of plexiglass (3 mm thick) and photocopied (Wilcox, 1982). For each seedling, the first-order lateral

TABLE I

Numerical values based on visual estimates (percentages) of the relative proportion of ectomycorrhiza of different morphological types formed by Pisolithus tinctorius on loblolly pine seedlings (Marx, 1990).

Ectomycorrhizal morphology

Visual estimate rating

Non-forked simple (%)

Coralloid <4 tips

(%)

Coralloid 4-10 tips (%)

Multiple coralloid fan-like

>10 tips (%)

1

100

0

0

0

2

50

50

0

0

3

25

75

0

0

4

25

50

25

0

5

25

25

25

25

6

0

25

50

25

7

0

25

25

50

8

0

0

50

50

9

0

0

25

75

10

0

0

0

100

roots are counted and the total length of lateral roots of all orders supporting short roots and ectomycorrhiza are then measured to the nearest 0.5 cm with a planimeter. From these and other measurements, the total number of short roots and ectomycorrhiza are derived for each seedling.

Photocopying of root systems, originally suggested by Wilcox (1982) not only provides excellent permanent records, it also permits accurate determination of lateral root numbers and their length. However, visual determinations of percentages of ectomycorrhiza and morphological types of ectomycorrhiza cannot be obtained from the photocopies. Many ectomycorrhiza are damaged or lost before photocopying when lateral roots are cut from the taproot, free water is removed from the lateral roots, and the lateral roots are arranged on the plastic sheets. Short roots, the number per cm of lateral root, are easily counted from a sample before photocopying and used to derive the total number of short roots per seedling after lateral root lengths are measured on photocopies. These data, along with visual estimates of percentage of ectomycorrhizal development, provide accurate estimates of the total number of ectomycorrhiza on each seedling. When differences in total lateral root length are small because of soil treatments, significant differences between visual percentage estimates and derived numbers are rarely found. Sometimes treatments affect Pt index differently from other measurements. The Pt index may be markedly different from other Pt measurements when root colonization by Pt and naturally occurring ectomycorrhiza are both very low (Table II). In this situation, Pt indices are usually high for the quantity of Pt ectomycorrhiza involved and, therefore, misleading. The total ectomycorrhizal rating, which combines numbers and relative size of ectomycorrhiza on a root system, is an excellent indicator of ectomycorrhizal fungus vigour or its amount on the root system. In past research, most ectomycorrhizal assessments dealt only with percentage of colonized short roots or bifurcated tips. Size of ectomycorrhiza was not measured or estimated. Total ectomycorrhizal rating, percentage of roots colonized, and the derived number of ectomycorrhiza can be used together to reflect more accurately ectomycorrhizal status of root systems.

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