Death of Root Tissues in Standing [live] and Felled

Recycling tree root components is important in sustaining the productivity of southern pine forests. Death of outer cortical tissues and mortality of short roots is ubiquitous in conifers. Affected tissues lose their starch grains and accumulate secondary products, such as tannins. In this study, lo-year-old loblolly pine trees were cut at the soil surface and sequential samples of roots were collected, fixed, embedded, and sectioned for light microscopy at monthly intervals. Observations showed roots of felled trees were similar to those of standing controls for approximately 5 months. Indicators of cell and tissue death were the disappearance of starch grains, increased tannin accumulation, and decreased staining of nuclei. This pattern of changes was remarkably similar to that of dying cortical cells. The long period (5 months) after felling and before the roots die probably has a significant effect on root microflora and the distribution of nutrients from the decomposition of surface woody debris and root systems. INTRODUCTION Loss of biomass from the crowns of loblolly pines (Pinus taeda L.) is easy to measure (Kozlowski and others 1991; Sampson and others 1998); loss of below-ground biomass can be harder to sample and measure (Kozlowski 1971, Ruark 1993). Death and rate of root decay are important on many reforestation sites where nutrient supply is marginal for seedling establishment and early tree growth. Nutrient cycling is influenced by how quickly root turnover occurs. Death of root cells was reviewed by Coulter as early as 1900 (Eames and MacDaniels 1947). I used ease of peeling the root cortex to assess the condition of primary roots. Smith (1935) Eames and MacDaniels (1947) and Esau (1953) detailed the function of the root cortex and its relationship to secondary growth. Those anatomical descriptions emphasize the complexity of below-ground biomass loss. Moreover, they suggest ttiat microscopical examination is essential for classifying cortical cells as dead. Medical investigators routinely use a number of cellular traits to determine cell death (Ellis and others 1991, Robbins 1987). Emphasis is placed on the condition of the nucleus when standardized stain schedules are applied to sections of tissue. I applied such schedules to pine root tissues. My objective was to devise quantitative measurements of cell traits that would precisely define root cell death. After accomplishing this, cell death was induced by tree felling and studied in detail. These two approaches provided a quantitative method for studying below-ground biomass in loblolly pine roots. SITES Observations to select methods and cellular traits were made on roots from young (5 to 10 years) loblolly pine stands in the Palustris Experimental Forest (Louisiana), the Homochitto National Forest (Mississippi), and in a Forest Service planting near Laurinburg, NC. A total of 5,476 roots were sectioned and stained for light microscopy., Experiments to induce root-cell death were conducted in the Palustris Experimental Forest. Treatments imposed in a 1 O-year-old loblolly pine study area included: (1) a control (no treatment), (2) felling in February and May 1994, (3) girdling at breast height, and (4) pruning lower limbs (leaving the top l/3 of crown). Root anatomy of 10 trees each of the control and those that were felled in February and May (treatment 2) was evaluated each month for 6 months. Treatments 3 and 4 were applied in May and sampled only 5 months following treatment. PROCEDURES Roots were sampled 1 m from the stem to a 20-cm depth for 6 to 10 trees at each site (Walkinshaw 1995). A cross-section of each root cl cm in diameter was excised and placed unwashed into formalin-acetic acid-alcohol (FAA) (Sass 1951). After 2 to 4 weeks, roots were rinsed with 70 percent ethyl alcohol. Specimens were cut to 1 to 3 mm, dehydrated in ethyl alcohol series, embedded in paraffin and cut into 7to IO-pm sections. Two or three sections that contained 9 to 18 roots from a single tree were mounted on a slide. Nine slides were prepared for each tree. Several staining schedules were used on root sections during the observation phase: acid fuchsin, Congo red, Giemsa, Groett’s methenamine, safranin-aniline blue, toluidine blue, hematoxylin-eosin, Papanicolaou’s schedule, and an acid&hi schedule (Haas 1980). Only the last three were used during the experimental phase. Root traits were scored as proportions or as real values. Papanicolaou’s schedule was read for two slides with two or three sets of roots per slide. I used hematoxylin-ecsin stain to verity nuclear viability in cells. Starch and tannin deposits were confirmed using an acid-Schiff schedule (Walkinshaw and Tiaras 1998). Cell traits used as dependent variables in the treatment evaluations are listed in table 1. RESULTS Initial Observations Shedding of the root cortex was first indicated in a large number of cells, distributed at random, by the breakdown of starch grains. Nuclei with changed stain affinity were prominent in most parenchyma and cortical ray cells. Cytoplasm became condensed to a small volume in the cortical cell periphery. These cells appeared net-like with primary cell walls held to each other and to the thin residual of living cortical cells. Nuclear staining as a measure of loss of vitality indicated that death of the cortex shed occurs after the loss of starch grains (Greenberg 1997). The proportion of roots with shedding was high in collections from 5and IO-year-old trees in the Palustris Experimental ’ Paper presented at the Tenth Biennial Southern Silvicultural Research Conference, Shreveport, LA, February 16-l 6, 1999. ’ Emeritus Scientist, USDA Forest Service, Southern Research Station, Pineville, LA 71360.